OB polypeptide antibodies and method of making

ABSTRACT

The present invention relates generally to the control of body weight of animals including mammals and humans, and more particularly to materials identified herein as modulators of body weight, and to diagnostic and therapeutic uses of such modulators. In its broadest aspect, the present invention relates to nucleotide sequences corresponding to the murine and human OB gene, and two isoforms thereof, and proteins expressed by such nucleotides or degenerate variations thereof, that demonstrate the ability to participate in the control of mammalian body weight and that have been postulated to play a critical role in the regulation of body weight and adiposity. The present invention further provides nucleic acid molecules for use as molecular probes or as primers for polymerase chain reaction (PCR) amplification. In further aspects, the present invention provides cloning vectors and mammalian expression vectors comprising the nucleic acid molecules of the invention. The invention further relates to host cells transfected or transformed with an appropriate expression vector and to their use in the preparation of the modulators of the invention. Also provided are antibodies to the OB polypeptide. Moreover, a method for modulating body weight of a mammal is provided.

The research leading to the present inventions was funded in part byGrant No. DK 41096 from the National Institutes of Health. Thegovernment may have certain rights in the invention.

RELATED APPLICATIONS

The present application is a continuation-in-part of copendingapplication Ser. No. 08/438,431, filed May 10, 1995, which in turn is acontinuation-in-part of copending application Ser. No. 08/347,563, filedNov. 30, 1994, which in turn is a continuation-in-part of copendingapplication Ser. No. 08/292,345, filed Aug. 17, 1994, to each of whichthe instant application claims the benefit of the filing date pursuantto 35 U.S.C. § 120, and each of which is incorporated herein byreference in its entirety.

TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to the control of body weight ofmammals including animals and humans, and more particularly to materialsidentified herein as modulators of weight, and to the diagnostic andtherapeutic uses to which such modulators may be put.

BACKGROUND OF THE INVENTION

Obesity, defined as an excess of body fat relative to lean body mass, isassociated with important psychological and medical morbidities, thelatter including hypertension, elevated blood lipids, and Type II ornon-insulin-dependent diabetes melitis (NIDDM). There are 6-10 millionindividuals with NIDDM in the U.S., including 18% of the population of65 years of age [Harris et al., Int. J. Obes., 11:275-283 (1987)].

Approximately 45% of males and 70% of females with NIDDM are obese, andtheir diabetes is substantially improved or eliminated by weightreduction [Harris, Diabetes Care, 14(3):639-648 (1991)]. As describedbelow, both obesity and NIDDM are strongly heritable, though thepredisposing genes have not been identified. The molecular genetic basisof these metabolically related disorders is an important, poorlyunderstood problem.

The assimilation, storage, and utilization of nutrient energy constitutea complex homeostatic system central to survival of metazoa. Amongland-dwelling mammals, storage in adipose tissue of large quantities ofmetabolic fuel as triglycerides is crucial for surviving periods of fooddeprivation. The need to maintain a fixed level of energy stores withoutcontinual alterations in the size and shape of the organism requires theachievement of a balance between energy intake and expenditure. However,the molecular mechanisms that regulate energy balance remain to beelucidated. The isolation of molecules that transduce nutritionalinformation and control energy balance will be critical to anunderstanding of the regulation of body weight in health and disease.

An individual's level of adiposity is, to a large extent, geneticallydetermined. Examination of the concordance rates of body weight andadiposity amongst mono- and dizygous twins or adoptees and theirbiological parents have suggested that the heritability of obesity(0.4-0.8) exceeds that of many other traits commonly thought to have asubstantial genetic component, such as schizophrenia, alcoholism, andatherosclerosis [Stunkard et al., N. Engi. J. Med., 322:1483-1487(1990)]. Familial similarities in rates of energy expenditure have alsobeen reported [Bogardus et al., Diabetes, 35:1-5 (1986)]. Geneticanalysis in geographically delimited populations has suggested that arelatively small number of genes may account for the 30-50% of variancein body composition [Moll et al., Am. J. Hum. Genet., 49:1243-1255(1991)]. However, none of the genes responsible for obesity in thegeneral population have been genetically mapped to a definitechromosomal location.

Rodent models of obesity include seven apparently single-gene mutations.The most intensively studied mouse obesity mutations are the ob (obese)and db (diabetes) genes. When present on the same genetic strainbackground, ob and db result in indistinguishable metabolic andbehavioral phenotypes, suggesting that these genes may function in thesame physiologic pathway [Coleman et al., Diabetologia, 14:141-148(1978)]. Mice homozygous for either mutation are hyperphagic andhypometabolic, leading to an obese phenotype that is notable at onemonth of age. The weight of these animals tends to stabilize at 60-70 g(compared with 30-35 g in control mice). ob and db animals manifest amyriad of other hormonal and metabolic changes that have made itdifficult to identify the primary defect attributable to the mutation[Bray et al., Am. J. Clin. Nutr., 50:891-902 (1989)].

Each of the rodent obesity models is accompanied by alterations incarbohydrate metabolism resembling those in Type II diabetes in man. Insome cases, the severity of the diabetes depends in part on thebackground mouse strain [Leiter, Endocrinology, 124:912-922 (1989)]. Forboth ob and db, congenic C57BL/Ks mice develop a severe diabetes withultimate β cell necrosis and islet atrophy, resulting in a relativeinsulinopenia. Conversely, congenic C57BL/6J ob and db mice develop atransient insulin-resistant diabetes that is eventually compensated by βcell hypertrophy resembling human Type II diabetes.

The phenotype of ob and db mice resembles human obesity in ways otherthan the development of diabetes--the mutant mice eat more and expendless energy than do lean controls (as do obese humans). This phenotypeis also quite similar to that seen in animals with lesions of theventromedial hypothalamus, which suggests that both mutations mayinterfere with the ability to properly integrate or respond tonutritional information within the central nervous system. Support forthis hypothesis comes from the results of parabiosis experiments[Coleman, Diabetologia, 9:294-298 (1973)] that suggest ob mice aredeficient in a circulating satiety factor and that db mice are resistantto the effects of the ob factor (possibly due to an ob receptor defect).These experiments have led to the conclusion that obesity in thesemutant mice may result from different defects in an afferent loop and/orintegrative center of the postulated feedback mechanism that controlsbody composition.

Using molecular and classical genetic markers, the ob and db genes havebeen mapped to proximal chromosome 6 and midchromosome 4, respectively[Bahary et al., Proc. Nat. Acad. Sci. USA, 87:8642-8646 (1990); Friedmanet al., Genomics, 11:1054-1062 (1991)]. In both cases, the mutations mapto regions of the mouse genome that are syntenic with human, suggestingthat, if there are human homologs of ob and db, they are likely to map,respectively, to human chromosomes 7q and 1p. Defects in the db gene mayresult in obesity in other mammalian species: in genetic crosses betweenZucker fa/fa rats and Brown Norway +/+ rats, the fa mutation (ratchromosome 5) is flanked by the same loci that flank db in mouse [Truettet al., Proc. Natl. Acad. Sci. USA, 88:7806-7809 (1991)].

Because of the myriad factors that seem to impact body weight, it hasnot been possible to predict which factors and, more particularly, whichhomeostatic mechanisms is actually primarily determinative. Nonetheless,the apparent connection between the ob gene and the extent andcharacteristics of obesity have prompted the further investigation andelucidation that is reflected by the present application. It is theidentification of the sequence of the gene and corresponding peptidematerials, to which the present invention following below directsitself.

The citation of any reference herein should not be construed as anadmission that such reference is prior art to the instant invention.Full citations of references cited by author and year are found at theend of the specification.

SUMMARY OF THE INVENTION

In its broadest aspect, the present invention relates to the elucidationand discovery of nucleic acids, and proteins putatively expressed bysuch nucleic acids or degenerate variants thereof, that demonstrate theability to participate in the control of mammalian body weight. Thenucleic acids in object represent the coding sequences corresponding tothe murine and human OB gene, that is postulated to play a critical rolein the regulation of body weight and adiposity. Data presented hereinindicates that the polypeptide product of the gene in question issecreted by the cells that express it and that the polypeptide functionsas a hormone.

In addition, the Examples herein demonstrate that the OB polypeptide,alternatively termed herein "leptin," circulates in mouse, rat, andhuman plasma. Leptin is absent in plasma from ob/ob mice, and is presentat ten-fold higher concentrations in plasma from db/db mice, andtwenty-fold higher concentrations in fa/fa rats. Most significantly,daily injections of recombinant leptin dramatically reduces the bodymass of ob/ob mice, significantly affects the body weight of wild-typemice, and has no effect on db/db mice.

In a further aspect, the OB polypeptide from one species is biologicallyactive in another species. In particular, the human OB polypeptide isactive in mice.

In a first instance, the modulators of the present invention comprisenucleic acid molecules, including recombinant DNA molecules (e.g., cDNAor a vector containing the cDNA or isolated genomic DNA) or cloned genes(i.e., isolated genomic DNA), or degenerate variants thereof, whichencode polypeptides themselves serving as modulators of weight controlas hereinafter defined, or conserved variants or fragments thereof,particularly such fragments lacking the signal peptide (alternativelyreferred to herein as mature OB polypeptide), which polypeptides possessamino acid sequences such as set forth in FIGS. 1A through E (SEQ IDNO:2), FIG. 3 (SEQ ID NO:4), FIG. 5 (SEQ ID NO:5) and FIG. 6 (SEQ IDNO:6). In specific embodiments, amino acid sequences for two variants ofmurine and human OB polypeptides are provided. Both polypeptides arefound in a form with glutamine 49 deleted, which may result from an mRNAsplicing anomaly. The OB polypeptides from various species may be highlyhomologous; as shown in FIG. 4, murine and human OB polypeptides aregreater than 80% homologous.

The nucleic acid molecules, recombinant DNA molecules, or cloned genes,may have the nucleotide sequences or may be complementary to DNA codingsequences shown in FIGS. 1A through E (SEQ ID NO:1) and FIGS. 2A and B(SEQ ID NO:3). In particular, such DNA molecules can be cDNA or genomicDNA isolated from the chromosome. Nucleic acid molecules of theinvention may also correspond to 5' and 3' flanking sequences of the DNAand intronic DNA sequences. Accordingly, the present invention alsorelates to the identification of a nucleic acid having a nucleotidesequence selected from the sequences of FIG. 1A through E (SEQ ID NO:1)and FIG. 2A and B (SEQ ID NO:3) herein, and degenerate variants, allelicvariations, and like cognate molecules.

A nucleic acid molecule of the invention can be DNA or RNA, includingsynthetic variants thereof having phosphate or phosphate analog, e.g.,thiophosphate, bonds. Both single-stranded and double-stranded sequencesare contemplated herein.

The present invention further provides nucleic acid molecules for use asmolecular probes, or as primers for polymerase chain reaction (PCR)amplification, i.e., synthetic or natural oligonucleotides having asequence corresponding to a portion of the sequences shown in FIGS. 1Athrough E (SEQ ID NO:1), FIGS. 2A and B (SEQ ID NO:3) and FIGS. 20Athrough C (SEQ ID NOS:22 and 24); or the 5' and 3' flanking sequences ofthe coding sequences; or intronic sequences of the genomic DNA. Inparticular, the invention contemplates a nucleic acid molecule having atleast about 10 nucleotides, wherein a sequence of the nucleic acidmolecule corresponds to a nucleotide sequence of the same number ofnucleotides in the nucleotide sequences of FIGS. 1A through E (SEQ IDNO:1), FIGS. 2A and B (SEQ ID NO:3) and FIGS. 20A through C (SEQ IDNOS:22 and 24), or a sequence complementary thereto. More preferably,the nucleic acid sequence of the molecule has at least 15 nucleotides.Most preferably, the nucleic acid sequence has at least 20 nucleotides.In an embodiment of the invention in which the oligonucleotide is aprobe, the oligonucleotide is detectably labeled, e.g., with aradionuclide (such as ³² P), or an enzyme.

In further aspects, the present invention provides a cloning vector,which comprises the nucleic acids of the invention that encode the OBpolypeptide; and a bacterial, insect, or a mammalian expression vector,which comprises the nucleic acid molecules of the invention encoding theOB polypeptide, operatively associated with an expression controlsequence. Accordingly, the invention further relates to a host cell,such as a bacterial cell, yeast cell, insect cell, or a mammalian cell,transfected or transformed with an appropriate expression vector, andcorrespondingly, to the use of the above mentioned constructs in thepreparation of the modulators of the invention.

In yet a further aspect, the present invention relates to antibodiesthat bind to the OB polypeptide. Such antibodies may be generatedagainst the full-length polypeptide, or antigenic fragments thereof. Inone aspect, such antibodies inhibit the functional (i.e., body weightand fat composition modulating) activity of the OB polypeptide. Inanother aspect, antibodies can be used to determine the level ofcirculating OB polypeptide in plasma or serum. In yet a further aspect,region-specific antibodies, particularly monoclonal antibodies, can beused as probes of OB polypeptide structure.

All of the foregoing materials are to be considered herein as modulatorsof body weight and fat composition, and as such, may be used in avariety of contexts. Specifically, the invention contemplates bothdiagnostic and therapeutic applications, as well as certain agriculturalapplications, all contingent upon the use of the modulators definedherein, including both nucleic acid molecules and peptides. Moreover,the modulation of body weight carries specific therapeutic implicationsand benefits, in that conditions where either obesity or, conversely,cachexia represent undesired bodily conditions, can be remedied by theadministration of one or more of the modulators of the presentinvention.

Thus, a method for modulating body weight of a mammal is proposed thatcomprises controlling the expression of the protein encoded by a nucleicacid having a nucleotide sequence selected from the sequence of FIG. 1Athrough E (SEQ ID NO:1), the sequence of FIG. 2A and B (SEQ ID NO:3) anddegenerate and allelic variants thereof. Such control may be effected bythe introduction of the nucleotides in question by gene therapy into fatcells of the patient or host to control or reduce obesity. Conversely,the preparation and administration of antagonists to the nucleotides,such as anti-sense molecules, would be indicated and pursued in theinstance where conditions involving excessive weight loss, such asanorexia nervosa, cancer, or AIDS are present and under treatment. Suchconstructs would be introduced in a similar fashion to the nucleotides,directly into fat cells to effect such changes.

Correspondingly, the proteins defmed by FIGS. 1A through E, 3, 5, and 6(SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6), conservedvariants, active fragments thereof, and cognate small molecules could beformulated for direct administration for therapeutic purposes, to effectreduction or control of excessive body fat or weight gain.Correspondingly, antibodies and other antagonists to the stated proteinmaterials, such as fragments thereof, could be prepared and similarlyadministered to achieve the converse effect. Accordingly, the inventionis advantageously directed to a pharmaceutical composition comprising anOB polypeptide of the invention, or alternatively an antagonist thereof,in an admixture with a pharmaceutically acceptable carrier or excipient.

In addition, the OB polypeptide of the invention may be administered forits cosmetic effects, e.g., to improve body appearance by reducing fatdeposits. The OB polypeptide can be used independently or in conjunctionwith other cosmetic strategies, e.g., surgery, for its cosmetic effects.

The diagnostic uses of the present nucleotides and correspondingpeptides extend to the use of the nucleic acids to identify frthermutations of alielic variations thereof, so as to develop a repertoireof active nucleotide materials useful in both diagnostic and therapeuticapplications. In particular, both homozygous and heterozygous mutationsof the nucleotides in question could be identified that would bepostulated to more precisely quantitate the condition of patients, todetermine the at-risk potential of individuals with regard to obesity.Specifically, heterozygous mutations are presently viewed as associatedwith mild to moderate obesity, while homozygous mutations would beassociated with a more pronounced and severe obese condition.Corresponding DNA testing could then be conducted utilizing theaforementioned ascertained materials as benchmarks, to facilitate anaccurate long term prognosis for particular tendencies, so as to be ableto prescribe changes in either dietary or other personal habits, ordirect therapeutic intervention, to avert such conditions.

The diagnostic utility of the present invention extends to methods formeasuring the presence and extent of the modulators of the invention incellular samples or biological extracts (or samples) taken from testsubjects, so that both the nucleic acids (genomic DNA or mRNA) and/orthe levels of protein in such test samples could be ascertained. Giventhat the increased activity of the nucleotide and presence of theresulting protein reflect the capability of the subject to inhibitobesity, the physician reviewing such results in an obese subject woulddetermine that a factor other than dysfunction with respect to thepresence and activity of the nucleotides of the present invention is acause of the obese condition. Conversely, depressed levels of thenucleotide and/or the expressed protein would suggest that such levelsmust be increased to treat such obese condition, and an appropriatetherapeutic regimen could then be implemented.

Further, the nucleotides discovered and presented in FIG. 1A through Eand FIG. 2A and B represent cDNA which, as stated briefly above, isuseful in the measurement of corresponding RNA. Likewise, recombinantprotein material corresponding to the polypeptides of FIGS. 1A through Eand 3 may be prepared and appropriately labeled, for use, for example,in radioimmunoassays, for example, for the purpose of measuring fatand/or plasma levels of the OB protein, or for detecting the presenceand level of a receptor for OB on tissues, such as the hypothalamus.

Yet further, the present invention contemplates not only theidentification of the nucleotides and corresponding proteins presentedherein, but the elucidation of the receptor to such materials. In suchcontext, the polypeptides of FIGS. 1A through E, 3, 5, and/or 6 could beprepared and utilized to screen an appropriate expression library toisolate active receptors. The receptor could thereafter be cloned, andthe receptor alone or in conjunction with the ligand could thereafter beutilized to screen for small molecules that may possess like activity tothe modulators herein.

Yet further, the present invention relates to pharmaceuticalcompositions that include certain of the modulators hereof, preferablythe polypeptides whose sequences are presented in SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:5 and SEQ ID NO:6, their antibodies, corresponding smallmolecule agonists or antagonists thereof, or active fragments preparedin formulations for a variety of modes of administration, where suchtherapy is appropriate. Such formulations would include pharmaceuticallyacceptable carriers, or other adjuvants as needed, and would be preparedin effective dosage ranges to be determined by the clinician or thephysician in each instance.

Accordingly, it is a principal object of the present invention toprovide modulators of body weight as defined herein in purified form,that exhibit certain characteristics and activities associated withcontrol and variation of adiposity and fat content of mammals. It is afurther object of the present invention to provide methods for thedetection and measurement of the modulators of weight control as setforth herein, as a means of the effective diagnosis and monitoring ofpathological conditions wherein the variation in level of suchmodulators is or may be a characterizing feature.

It is a still further object of the present invention to provide amethod and associated assay system for the screening of substances, suchas drugs, agents and the like, that are potentially effective to eithermimic or inhibit the activity of the modulators of the invention inmammals.

It is a still further object of the present invention to provide amethod for the treatment of mammals to control body weight and fatcontent in mammals, and/or to treat certain of the pathologicalconditions of which abnormal depression or elevation of body weight is acharacterizing feature.

It is a still further object of the present invention to prepare geneticconstructs for use in genetic therapeutic protocols and/orpharmaceutical compositions for comparable therapeutic methods, whichcomprise or are based upon one or more of the modulators, bindingpartners, or agents that may control their production, or that may mimicor antagonize their activities.

Other objects and advantages will become apparent to those skilled inthe art from a review of the ensuing description which proceeds withreference to the following illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A through E depicts the nucleic acid sequence (SEQ ID NO:1) anddeduced amino acid sequence (SEQ ID NO:2) derived for the murine OBcDNA. A 39 base pair 5' leader was followed by a predicted 167 aminoacid open reading frame and an approximately 3.7 kb 3' untranslatedsequence. (In previously filed application Ser. No. 08/347,563 filedNov. 30, 1994 and Ser. No. 08/438,431, filed May 10, 1995, an additional58-base 5' non-coding sequence was determined subsequently, to be acloning artifact. This artifact has no bearing on the coding region, the39 base 5' noncoding region presently depicted in FIG. 1, or 3'non-coding region of the gene.) A total of about 2500 base pairs of the3' untranslated sequence is shown. Analysis of the predicted proteinsequence by observation and using the SigSeq computer program indicatesthe presence of a signal sequence (underlined). Microheterogeneity ofthe cDNA was noted in that approximately 70% of the cDNAs had aglutamine codon at codon 49 and 30% did not (see FIGS. 5 and 6, infra).This amino acid is underlined, as is the arginine codon that is mutatedin C57BL/6J ob/ob mice (1J mice).

FIG. 2A and B depicts the nucleic acid sequence (SEQ ID NO:3) derivedfor the human OB cDNA. The nucleotides are numbered from 1 to 701 with astart site at nucleotide 46 and a termination at nucleotide 550.

FIG. 3 depicts the full deduced amino acid sequence (SEQ ID NO:4)derived for the human OB gene corresponding to the nucleic acid sequenceof FIG. 2A and B. The amino acids are numbered from 1 to 167. A signalsequence cleavage site is located after amino acid 21 (Ala) so that themature protein extends from amino acid 22 (Val) to amino acid 167 (Cys).

FIG. 4 depicts the comparison between the murine (SEQ ID NO:2) and human(SEQ ID NO:4) deduced amino acid sequences. The sequence of the human OBdeduced amino acid sequence was highly homologous to that of mouse.Conservative changes are noted by a dash, and non-conservative changesby an asterisk. The variable glutamine codon is underlined, as is theposition of the nonsense mutation in C57BL/6J ob/ob (1J) mice. Overall,there is 83% identity at the amino acid level, although only eightsubstitutions were found between the valine at codon 22 (immediatelydownstream of the signal sequence) and the cysteine at position 117.

FIG. 5 depicts the full length amino acid sequence (SEQ ID NO:5) derivedfor the murine OB gene as shown in FIG. 3, but lacking glutamine atposition 49. The amino acids are numbered from 1 to 166. A signalsequence cleavage site is located after amino acid 21 (Ala) (and thus,before the glutamine 49 deletion) so that the mature protein extendsfrom amino acid 22 (Val) to amino acid 166 (Cys).

FIG. 6 depicts the full deduced amino acid sequence (SEQ ID NO:6)derived for the human OB gene as shown in FIG. 4, but lacking glutamineat position 49. The amino acids are numbered from 1 to 166. A signalsequence cleavage site is located after amino acid 21 (Ala) (and thus,before the glutamine 49 deletion) so that the mature protein extendsfrom amino acid 22 (Val) to amino acid 166 (Cys).

FIG. 7 (A) Physical map of the location of OB in the murine chromosome,and the YAC and P1 cloning maps. "M and N" corresponds to Mull and NotIrestriction sites. The numbers correspond to individual animals thatwere recombinant in the region of OB of the 1606 meioses that werescored. Met, Pax 4, D6Rck39, D6Rck13, and Cpa refer to locations in theregion of OB that bind to the DNA probes. YACs were isolated usingD6Rck13 and Pax-4 as probes, and the ends were recovered usingvectorette PCR and/or plasmid end rescue and used in turn to isolate newYACs. (B) The resulting YAC contig. One of the YACs in this contig,Y902A0925, was chimeric. Each of the probes used to genotype therecombinant animals is indicated in parentheses. (6) Corresponds to YAC107; (5) corresponds to M16(+) (or M16(pLUS)); (4) corresponds toadu(+); (3) corresponds to aad(pICL); (2) corresponds to 53(pICL); and(1) corresponds to 53(+). (C) The P1 contig of bacteriophage P1 clonesisolated with selected YAC end probes. The OB gene was isolated in a P1clone isolated using the distal end of YAC YB6S2F12 (end (4))(alternatively termed herein adu(+)).

FIG. 8 presents a photograph of an ethidium bromide stain of 192independent isolates of the fourth exon trapping experiment that werePCR amplified and characterized.

FIG. 9 is a photograph of an ethidium bromide stain of PCR-amplifiedclones suspected of carrying OB. Each of the 7 clones that did not carrythe artifact was reamplified using PCR and electrophoresed on a 1%agarose gel in TBE and stained with ethidium bromide. The size markers(far left unnumbered lane) are the commercially available "1 kB ladder".Lane 1--clone 1D12, containing an "HIV sequence." Lane 2--clone 1F1, anovel clone outside of the OB region. Lane 3--clone 1H3. Lane 4--clone2B2, which is the identical to 1F1. Lane 5--clone 2G7, which contains anOB exon. Lane 6--clone 2G11, which is identical to 1F1. Lane 7--clone2H1, which does not contain an insert.

FIG. 10 presents the sequence of the 2G7 clone (SEQ ID NO:7), whichincludes an exon coding for a part of the OB gene. The primer sequencesused to amplify this exon are boxed in the figure (SEQ ID NOS:8 and 9).

FIG. 11 (A) Reverse transcription-PCR analysis of mRNA from differenttissues of the same mouse with the 2G7 primers and actin primers. TheRT-PCR reactions were performed using 100 ng of total RNA reversetranscribed with oligo dT as a primer for first strand cDNA synthesis.PCR amplification was performed for 35 cycles with 94° denaturation for1'; 55° hybridization for 1'; and 72° C. extensions for 2' with a 1'second autoextension per cycle. RT-PCR products were resolved in a 2%low melting point agarose gel run in 1× TBE buffer. (B) Northern blot ofmRNA from different organs of the mouse using PCR labeled 2G7 as aprobe. Ten μg of total RNA from each of the tissues was electrophoresedon an agarose gel with formaldehyde. The probe was hybridized at 65° C.in Rapid Hybe (Amersham). Autoradiographic signals were apparent after 1hour of exposure; the experiment shown was the result of a 24 hourexposure.

FIG. 12 (A) An ethidium bromide stain from an RT-PCR reaction on fatcell (white adipose tissue) RNA from each of the mouse strains listed.Total RNA (100 ng) for each sample was reverse transcribed using oligodT and reverse transcriptase, and the resulting single-stranded cDNA wasPCR amplified with the 2G7 primers (lower bands) or actin primers (upperbands). Both the 2G7 and actin primers were included in the same PCRreaction. The products were run on a 1% agarose TBE gel. (B) Northernanalysis corresponding to (A). Ten μg of fat cell (white adipose tissue)RNA from each of the strains indicated were run out and probed with thePCR labeled 2G7 probe as in FIG. 11B, above. An approximately 20-foldincrease in the level of 2G7 mRNA was apparent in white fat RNA from theC57BL/6J ob/ob (1J) strain relative to lean littermates. In both theRT-PCR and Northern experiments there was no detectable signal in 2G7RNA from the SM/Ckc-+^(Dac) ob^(2J) /ob^(2J) (2J) mice even after a 2week exposure. A 24 hour autoradiographic exposure is shown. The samefilter was hybridized to an actin probe (bottom portion of the panel).

FIG. 13 is a Northern analysis of additional 2J animals and controlanimals that confirms the absence of the ob mRNA from 2J animals. TheNorthern analysis was performed as in FIGS. 11 and 12. In this case, thecontrol RNA was ap2, a fat specific transcript. There is no significanceto the varying density of the ap2 bands.

FIG. 14 compares the DNA sequence of the C57BL/6J (normal) and theC57BL/6J ob/ob (1J) mice in the region of the point mutation that leadsto introduction of a premature stop codon (nonsense mutation) in themutant strain cDNA. The ob/ob mice had a C→T mutation that changed anarginine residue at position 105. This base change is shown as theoutput from the automated DNA sequencer. RT-PCR was performed usingwhite fat RNA from both strains (+/+ and oblob) using primers from the5' and 3' untranslated regions. The PCR reaction products were gelpurified and directly sequenced manually and using an AppliedBiosystems, Inc. 373A automated sequencer with primers along bothstrands of the coding sequence.

FIG. 15 (A) Southern blot of genomic DNA from each of the mouse strainslisted. Approximately 5 μg of DNA (derived from genomic DNA preparedfrom liver, kidney or spleen) was restriction digested with therestriction enzyme indicated. The DNA was then electrophoresed in a 1%agarose TBE gel and probed with PCR labeled 2G7. Restriction digestionwith BglII revealed an increase in the size of an approximately 9 kB(the largest) BglII fragment in SM/Ckc-+^(Dac) ob^(2J) /ob^(2J) (2J)DNA. RFLPs were not detectable with any other restriction enzymes.Preliminary restriction mapping of genomic DNA indicated that thepolymorphic BglII site is about 7 kB upstream of the transcription startsite. None of the other enzymes tested extend past the mRNA start site.(B) Segregation of a BglII polymorphism in the SM/Ckc-+^(Dac) ob^(2J)/ob^(2J) strain. Six obese and five lean progeny from the samegeneration of the coisogenic SM/Ckc-+^(Dac) ob^(2J) /ob^(2J) (2J) colonywere genotyped by scoring the BglII polymorphism as shown in (A). All ofthe phenotypically obese animals were homozygous for the larger alleleof the polymorphic BglII fragment. The DNA in the "control" lane wasprepared from an unrelated SM/Ckc-+^(Dac) +/+ mouse, bred separatelyfrom the SM/Ckc-+^(Dac) ob^(2J) /ob^(2J) colony.

FIG. 16 is a Southern blot of EcoRI digested genomic DNA from thespecies listed, using an OB cDNA as a probe (i.e., a zoo blot).Hybridization signals were detectable in every vertebrate sample, evenafter a moderate stringency hybridization. The cat DNA in thisexperiment was slightly degraded. The restricted DNA was run on a 1%agarose TBE gel, and transferred to an imobilon membrane for probing.The filter was hybridized at 65° C. and washed in 2X SSC/0.2% SDS at 65°C. twice for twenty minutes and exposed for 3 days using Kodak(Rochester, N.Y.) X-OMAT film.

FIG. 17 presents the expression cloning region of vector pET-15b(Novagen) (SEQ ID NOS.:11 and 12).

FIG. 18 presents analysis of the eluate from a His-binding resin (Ni)column for a recombinant mature murine OB fusion to a His-tag (A) andmature human OB fusion to a His-tag (B). Bacteria were transformed withvectors pETM9 and pETH14, respectively. Upon induction with 1 mM IPTG atoptimal conditions, the transformed bacteria were able to produce100-300 μg/ml of OB fusion protein, primarily in the inclusion bodies.The inclusion bodies were solubilized with 6M guanidine-HCl or urea, andfusion protein (present in the lysis supernatant) was loaded on theHis-binding resin (Ni) column in 10 ml of 1x binding buffer with urea.The column was eluted stepwise with 5 ml aliquots of 20 μM, 60 μM, and300 μM imidazole, and finally with strip buffer. The aliquots wereanalyzed for the presence of OB polypeptide fusion on a 15% acrylamidegel. Each lane contains the equivalent of 100 μl of bacterial extract.

FIG. 19 (A) In vitro translation of OB RNA. A human OB cDNA wassubcloned into the pGEM vector. The plasmid was linearized and plusstrand RNA was synthesized using Sp6 polymerase. The in vitrosynthesized RNA was translated in the presence or absence of caninepancreatic microsomal membranes. An approximately 18 kD primarytranslation product was seen after in vitro translation. The addition ofmicrosomal membranes to the reaction led to the appearance of a secondtranslation product about 2 kD smaller than the primary translationproduct. The size of the translation product of interleukin-1α RNA,which lacks an encoded signal sequence, was unchanged by the addition ofmicrosomal membranes. These data indicated the presence of a functionalsignal sequence. (B) In vitro translation in the presence or absence ofproteinase K. Protease treatment resulted in complete proteolysis of the18 kD primary translation product, while the 16 kD processed form wasunaffected. Permeabilization of the microsome with 0.1% TRITON-X100rendered the processed form protease sensitive. These results indicatethat the product had translated into the lumen of the microsome.

FIG. 20 (A through E) The sequence of the human OB gene (SEQ ID NOS:22and 24). (F) A schematic diagram of the murine OB gene. (G) A schematicdiagram of the human OB gene. In both (F) and (G), the start and stopcodons are underlined. There is no evidence of a first intron homologousto the mouse first intron in the human gene, but its existence cannot beexcluded.

FIG. 21 presents a schematic drawing of one of the cloning strategiesemployed to achieve recombinant expression of OB in Pichia yeast. (A)Expression vector of OB with an α-mating factor signal sequence. (B)Schematic drawing of the structure of the recombinant fusion protein,including the amino acid sequence (SEQ ID NO:26) showing the XhoI siteand putative KEX-2 and STE-13 cleavage sites, and the N-terminal surplusamino acids present after KEX-2 cleavage (SEQ ID NO:27). (C) Analternative strategy for producing mature OB involves preparing aconstruct with an amino acid sequence corresponding to a XhoI cleavagesite and a KEX-2 cleavage site immediately upstream of the mature OBpolypeptide sequence (SEQ ID NO:28).

FIG. 22 Alternative expression strategy in Pichia. (A) Expression vectorof an OB fusion with a His-tag adopted from the pET expression systemunder control of the α-mating factor signal sequence (SEQ ID NO:33). (B)Schematic drawing of the structure of the recombinant OB fusion proteincontaining a His-tag, which includes the α-matipg factor signalsequence, putative KEX-2 and STE-13 cleavage sites, the His-tag, and athrombin cleavage site, which would yield OB with three surplusN-terminal amino acid residues.

FIG. 23 (A) PAGE analysis of expression of murine OB (both themicroheterogenous forms, i.e., containing and missing Gln 49) intransformed Pichia yeast. The expected band of approximately 16 kD isvisible in the transformed yeast culture fluid (second and third lanes),but not in culture fluid from non-transformed yeast (first lane). (B)PAGE analysis of partially purified recombinant OB polypeptide oncarboxymethyl cellulose, a weak cation exchanger. A band of about 16 kDis very visible in fractions 3 and 4 from the column, which was elutedwith 250 mM NaCl. Lane 1--loaded sample; lane 2--flow through; lanes 3-5--fractions eluted with 250 mM NaCl.

FIG. 24 shows that the OB protein circulates in mouse plasma. (A)Immunoprecipitations from mouse blood. 0.5 ml of mouse plasma waspre-cleared with unconjugated sepharose and incubated overnight withimmunopurified anti-OB antibodies conjugated to sepharose 4B beads. Theirnmunoprecipitate was separated on a 15% SDS-PAGE gel, transferred andWestern blotted with an anti-OB antibody. The protein migrated with amolecular weight of approximately 16 kD, to the same position as themature mouse OB protein expressed in yeast. The protein was absent inplasma from C57BL/6J ob/ob mice and increased ten-fold in plasma fromC57BLB/Ks db/db mice relative to wild type mice. db mice have beensuggested to overproduce the OB protein, secondary to resistance to itseffects. (B) Increased levels of OB in fatty rats. The fatty rat isobese as a result of a recessive mutation on rat chromosome 5. Geneticdata has suggested a defect in the same gene mutated in db mice. Plasmafrom fatty rats and lean littermates was immunoprecipitated and run onWestern blots. A twenty-fold increase in the circulating level of OB isseen in the mutant animals. (C). Quantitation of the OB protein in mouseplasma. Increasing amounts of the recombinant mouse protein were addedto 100λ of plasma from ob mice and immunoprecipitated. The signalintensity on Western blots was compared to that from 100λ of plasma fromwild-type mice. A linear increase in signal intensity was seen withincreasing amounts of recombinant protein demonstrating that theimmunoprecipitations were performed under conditions of antibody excess.Similar signals were seen in the wild-type plasma sample and the samplewith 2 ng of recombinant protein indicating the circulating level inmouse plasma is approximately 20 ng/ml. (D) OB protein in adipose tissueextracts. Cytoplasmic extracts of mouse adipose tissue were preparedfrom db and wild-type mice. Western blots showed increased levels of the16 kD protein in extracts prepared from db mice.

FIG. 25 shows that the OB protein circulates at variable levels in humanplasma. (A) Western blots of human plasma. Plasma samples were obtainedfrom six lean volunteers. Immunoprecipitation and Western blottingrevealed the presence of an immunoreactive 16 kD protein, identical insize to a recombinant 146 amino acid human protein expressed in yeast.Variable levels of the protein were seen in each of the six samples. (B)An ELISA (Enzyme Linked Immunoassay) for human OB. Microtiter plateswere coated with immunopurified anti-human OB antibodies. Known amountsof recombinant protein were added to the plates and detected usingimmunopurified biotinylated anti-OB antibodies. Absorbance at 414 nm wasplotted against known concentrations of OB to yield a standard curve.The resulting standard curve showed that the assay was capable ofdetecting 1 ng/ml or more of the human OB protein. (C) Quantitation ofthe OB protein in human plasma. An ELISA immunoassay was performed using100λ of plasma from the six lean volunteers and the standards used inpanel B. Levels of the OB protein ranging from 2 ng/ml in HP1 to 15ng/ml in HP6 were seen. These data correlated with the Western blot datain panel A.

FIG. 26 shows that the OB protein forms inter- or intramoleculardisulphide bonds. (A) Western blots under reducing and non-reducingconditions. The Western blots of mouse and human plasma were repeatedwith and without the addition of reducing agents to the sample buffer.When β-mercaptoethanol is omitted from the sample buffer,immunoprecipitates from db plasma migrate with an apparent molecularmass of 16 kD and 32 kD. Addition of β-mercaptoethanol to the bufferleads to the disappearance of the 32 kD moiety (see FIG. 24). Thisresult is recapitulated when the mouse protein is expressed in theyeast, Pichia pastoris. In this case, the mouse OB protein migrates tothe position of a dimer. Under reducing conditions the purifiedrecombinant mouse protein migrates with an apparent molecular weight of16 kD, indicating that the 32 kD molecular form is the result of one ortwo intermolecular disuphide bonds. The human protein expressed in vivoand in Pichia pastoris migrates with a molecular mass of 16 kD underboth reducing and non-reducing conditions (data not shown). (B) Thehuman protein expressed in yeast contains an intramolecular disulphidebond. Secreted proteins generally assume their correct conformation whenexpressed in the Pichia pastoris expression system. The 146 amino acidmature human protein was expressed in Pichia pastoris and purified fromthe yeast media by a two-step purification protocol involving IMAC andgel filtration. The purified recombinant protein was subjected to massspectrometry before and after cyanogen bromide cleavage. Cyanogenbromide cleaves at the carboxy terminus of methionine residues. Themolecular mass of the recombinant yeast protein was 16,024±3 Da(calculated molecular mass=16,024 Da). Cyanogen bromide cleaves afterthe three methionines in the protein sequence at amino acids 75, 89, and157. The cyanogen bromide fragment with measured mass 8435.6 Dacorresponds to amino acids 90-157 and 158-167 joined by a disulphidelinkage between cys-117 and cys-167 (calculated molecular mass=8434.5Da). N.D.=note detected.

FIG. 27 depicts the preparation of the bioactive recombinant protein.The nucleotide sequence corresponding to the 145 amino acid mature mouseOB protein was cloned into the pET 15b expression vector. This pETvector inserts a polyhistidine tract (His-tag) upstream of the clonedsequence which allows efficient purification using Immobilized MetalAffinity Chromatography (IMAC). The recombinant bacterial proteininitially partitioned in the insoluble membrane fraction after bacteriallysis. The membrane fraction was solubilized using guanidiumhydrochloride and loaded onto an IMAC column. The protein was elutedstepwise with increasing concentrations of imidazole as shown. Theeluted protein was refolded and treated with thrombin to remove theHis-tag, as described below. The final yield of soluble protein was 45ng/ml of bacterial culture.

FIG. 28 shows the biologic effects of the OB protein. Time course offood intake (panels A-C) and body weight (panels D-F). Groups of tenanimals received either daily intraperitoneal injections of the OBprotein at a dose of 5 mg/kg/day (solid squares), daily injections ofPBS (solid circles) or no treatment (solid triangles). The treatmentgroups included C57B1/6J ob/ob mice (panels A and D), C57Bl/Ks db/dbmice (panels B and E) and CBA/J+/+ mice (panels C and F). The foodintake of the mice was measured daily and the body weight was recordedat three to four day intervals as indicated. (The scale of the bodyweight in grams is different for the wild-type mice vs. the ob and dbmice.) The food intake of the ob mice receiving protein was reducedafter the first injection and stabilized after the fourth day at a levelapproximately 40% of that seen in the sham injected group (p<0.001). Thebody weight of these animals decreased an average of 1.3 grams/day andstabilized after three weeks to a level approximately 60% of thestarting weight (p<0.0001). No effect of the protein was demonstrable indb mice. Small but significant effects on body weight were observed inCBA/J mice at two early time points (p<0.02). The standard error of eachmeasure is depicted by a bar and the statistical significance of theseresults is shown in Table 1.

FIG. 29 shows the results of pair feeding of ob mice. (A) A group offour C57Bl/6J ob/ob mice were fed an amount of food equal to thatconsumed by the group of ob mice receiving recombinant protein. Theweight loss for both groups was calculated after five, eight, and twelvedays. The food-restricted mice lost (hatched bar) less weight than theob mice receiving protein (solid bar) (p<0.02). This result indicatesthat the weight-reducing effect of the OB protein is the result ofeffects on both food intake and energy expenditure. (B) Photograph of atreated ob mouse. Shown are two C57Bl/6J ob/ob mice. The mouse on theleft received PBS and weighed 65 grams, which was the starting weight.The mouse on the right received daily injections of the recombinant OBprotein. The starting weight of this animal was also 65 grams, and theweight after three weeks of protein treatment was 38 grams. (C) Liversfrom treated and untreated ob mice. Shown are livers from treated anduntreated C57Bl/6J ob/ob mice. The liver from the mouse receiving PBShad the gross appearance of a fatty liver and weighed 5.04 grams. Theliver from the mouse receiving the recombinant ob protein had a normalappearance and weighed 2.23 grams.

FIG. 30 shows the in situ hybridization of ob to adipose tissue. Senseand antisense ob RNA was labeled in vitro using Sp6 and T7 polymeraseand digoxigenin. The labeled RNAs were hybridized to paraffin embeddedsections of adipose tissue from epididymal fat pads of eight week oldC57Bl/Ks mice (labeled wild-type) and C57Bl/Ks db/db mice (labeled db).In the figure, the lipid droplets appear as unstained vacuoles withincells. The cytoplasm is a thin rim at the periphery of the cells and isindistinguishable from the cell membrane. Hybridization to all theadipocytes in the field was detected in the wild-type sections onlyusing the antisense probe and greatly increased levels were seen in thetissue sections from the db/db animals.

FIG. 31 shows that OB RNA is expressed in adipocytes in vivo and invitro. Total RNA (10 micrograms) from several different sources waselectophoresed on blotted and hybridized to an OB probe. Firstly,differences in cell buoyancy after collagenase digestion was used topurify adipocytes. OB RNA was present only in the adipocyte fraction.Lane S indicates the stromovascular fraction and A indicates theadipocyte fraction. In addition, OB RNA was not expressed in theundifferentiated 3T3-442 preadipocyte cells lane U. Differentiatedadipocytes from these cell lines expressed clearly detectable levels ofOB mRNA (lane D).

FIG. 32 shows that OB RNA is expressed in all adipose tissue depots. Allof the adipose tissue depots tested expressed OB RNA. The inguinal fatpad expressed somewhat lower RNA levels, although there was variabilityin the level of signals in different experiments. (FIG. 31A) Lanes (1)epididymal (2) inguinal (3) abdominal (4) parametrial fat pads. Brownfat also expressed a low level of OB RNA. (FIG. 31B) The level of OBexpression in brown fat was unchanged in animals housed at 4° C. for oneweek while the abundance of the brown fat specific UCP RNA, known to becold inducible, increased five-fold.

FIG. 33 depicts the expression of OB RNA in db/db and gold thioglucosetreated mice. Total RNA from the parametrial fat pads of goldthioglucose (GTG) and db/db treated mice was electrophoresed andNorthern blotted. GTG administered as a single dose is known to causeobesity by inducing specific hypothalamic lesions. (A) One month old CBAfemale mice were treated with GTG (0.2 mg/g), with a resulting increaseof >20 g in treated animals relative to control animals (<5 g). (B)Hybridization of an OB probe to RNA from dbldb and GTG treated micerevealed a twenty-fold increase in the abundance of OB RNA relative tocontrol RNA (actin or GAPDH).

FIG. 34 represents a Northern blot analysis of human RNA. Northern blotscontaining 10 mg of total RNA from human adipose tissue (FAT, panel A)and 2 mg of polyA+ RNA from other human tissues (panel B) werehybridized to human OB or human β-actin probes as indicated. An intensesignal at approximately 4.5 kb was seen with the adipose tissue totalRNA. Hybridization to the polyA+ RNA revealed detectable signals inheart (HE) and placenta (PL), whereas OB RNA was not detected in brain(BR), lung (LU), liver (LI), skeletal muscle (SM), kidney (KI), andpancreas (PA). In each case, the length of the autoradiographic exposureis indicated. Of note, the genesis of the lower molecular bands seen inplacental RNA (e.g., alternate splicing, RNA degradation) is not known.

FIG. 35 represents YAC contig containing the human OB gene and 8microsatellite markers. The YAC-based STS-content map of the region ofchromosome 7 containing the human OB gene is depicted, as deduced bySEGMAP/Version 3.29 [Green et al., PCR Methods Applic., 1:77-90 (1991)].The 19 uniquely-ordered STSs (see Table 3) are listed along the top. The8 microsatellite-specific STSs are indicated with stars (see Table 4).Also indicated are the STSs corresponding to the Pax4 and OB genes aswell as the predicted positions of the centromere (CEN) and 7q telomere(TEL) relative to the contig. Each of the 43 YAC clones is depicted by ahorizontal bar, with its name given to the left and estimated YAC size(in kb, measured by pulsed-field gel electrophoresis) provided inparenthesis. The presence of an STS in a YAC is indicated by a darkenedcircle at the appropriate position. When an STS corresponds to theinsert end of a YAC, a square is placed around the corresponding circle,both along the top (near the STS name) and at the end of the YAC fromwhich it was derived. For the 5 YACs at the bottom (below the horizontaldashed line), I or more STS(s) expected to be present (based on theestablished STS order) was not detected (as assessed by testing theindividual YACs with the corresponding STS-specific PCR assay(s) atleast twice), and these are depicted as open circles at the appropriatepositions. Most of the YACs were isolated from a human-hamster hybridcell-derived library [Green et al., Genomics, 25:170-183 (1995)], withtheir original names as indicated. The remaining YACs were isolated fromtotal human genomic libraries, and their original library locations areprovided in Table 3. Boxes are placed around the names of the 3 YACs(yWSS691, yWSS999, and yWSS2935) that were found by FISH analysis to mapto 7q31.3. The contig is displayed in its `uncomputed` form, where YACsizes are not used to estimate clone overlaps or STS spacing, and all ofthe STSs are therefore spaced in an equidistant fashion. In the`computed` form, where YAC sizes are used to estimate the relativedistance separating each pair of adjacent STSs as well as the extent ofclone overlaps, the total YAC contig appears to span just over 2 Mb.

DETAILED DESCRIPTION

The present invention relates to the elucidation and discovery of aprotein, termed herein OB polypeptide or leptin, nucleic acids encodingthe protein, including degenerate variations thereof, e.g., thatincorporate optimal codons for expression in a particular expressionsystem, which protein demonstrates the ability to participate in thecontrol of mammalian body weight. The nucleic acids in object representthe coding sequences corresponding to the murine and human OBpolypeptide, which is postulated to play a critical role in theregulation of body weight and adiposity. Data presented herein indicatethat the polypeptide product of a nuceic acid of the invention issecreted by the cells that express it, and that the polypeptidefunctions as a hormone. Additional experimental data demonstrate thatthe OB polypeptide is very effective in treating obesity in micecarrying a mutation of the ob gene. In addition, high bolus doses ormoderate continuous doses of OB polypeptide effect weight reduction innormal (wild-type) mice.

In addition, the Examples herein demonstrate that the OB polypeptide,alternatively termed herein "leptin," circulates in mouse, rat, andhuman plasma. Leptin is absent in plasma from ob/ob mice, and is presentat ten-fold higher concentrations in plasma from db/db mice, andtwenty-fold higher concentrations in fa/fa rats. Most significantly,daily injections of recombinant leptin dramatically reduce the body massof ob/ob mice, significantly affects the body weight of wild-type mice,and has no effect on db/db mice.

In a further aspect, the OB polypeptide from one species is biologicallyactive in another species. In particular, the human OB polypeptide isactive in mice.

In its primary aspect, the present invention is directed to theidentification of materials that function as modulators of mammalianbody weight. In particular, the invention concerns the isolation,purification and sequencing of certain nucleic acids that correspond tothe OB gene or its coding region in both mice and humans, as well as thecorresponding polypeptides expressed by these nucleic acids. Theinvention thus comprises the discovery of nucleic acids having thenucleotide sequences set forth in FIG. 1A through E (SEQ ID NO:1) andFIG. 2A and B (SEQ ID NO:3), and to degenerate variants, alleles andfragments thereof, all possessing the activity of modulating body weightand adiposity. The correspondence of the present nucleic acids to the OBgene portends their significant impact on conditions such as obesity aswell as other maladies and dysfunctions where abnormalities in bodyweight are a contributory factor. The invention extends to the proteinsexpressed by the nucleic acids of the invention, and particularly tothose proteins set forth in FIG. 1A through E (SEQ ID NO:2), FIG. 3 (SEQID NO:4), FIG. 5 (SEQ ID NO:5), and FIG. 6 (SEQ ID NO:6), as well as toconserved variants, active fragments, and cognate small molecules.

As discussed earlier, the weight control modulator peptides or theirbinding partners or other ligands or agents exhibiting either mimicry orantagonism to them or control over their production, may be prepared inpharmaceutical compositions, with a suitable carrier and at a strengtheffective for administration by various means to a patient experiencingabnormal fluctuations in body weight or adiposity, either alone or aspart of an adverse medical condition such as cancer or AIDS, for thetreatment thereof. A variety of administrative techniques may beutilized, among them oral administration, nasal and other forms oftransmucosal administration, parenteral techniques such as subcutaneous,intravenous and intraperitoneal injections, catheterizations and thelike. Average quantities of the recognition factors or their subunitsmay vary and in particular should be based upon the recommendations andprescription of a qualified physician or veterinarian.

In accordance with the above, an assay system for screening potentialdrugs effective to mimic or antagonize the activity of the weightmodulator may be prepared. The weight modulator may be introduced into atest system, and the prospective drug may also be introduced into theresulting cell culture, and the culture thereafter examined to observeany changes in the activity of the cells, due either to the addition ofthe prospective drug alone, or due to the effect of added quantities ofthe known weight modulator.

As stated earlier, the molecular cloning of the OB gene described hereinhas led to the identification of a class of materials that function onthe molecular level to modulate mammalian body weight. The discovery ofthe modulators of the invention has important implications for thediagnosis and treatment of nutritional disorders including, but notlimited to, obesity, weight loss associated with cancer and thetreatment of diseases associated with obesity such as hypertension,heart disease, and Type II diabetes. In addition, there are potentialagricultural uses for the gene product in cases where one might wish tomodulate the body weight of domestic animals. Finally, to the extentthat one or more of the modulators of the invention are secretedmolecules, they can be used biochemically to isolate their receptorusing the technology of expression cloning. The discussion that followswith specific reference to the OB gene bears general applicability tothe class of modulators that comprise a part of the present invention,and is therefore to be accorded such latitude and scope ofinterpretation.

As noted above, the functional activity of the OB polypeptide can beevaluated transgenically. In this respect, a transgenic mouse model canbe used. The ob gene can be used in complementation studies employingtransgenic mice. Transgenic vectors, including viral vectors, or cosmidclones (or phage clones) corresponding to the wild-type locus ofcandidate gene, can be constructed using the isolated OB gene. Cosmidsmay be introduced into transgenic mice using published procedures[Jaenisch, Science, 240:1468-1474 (1988)]. The constructs are introducedinto fertilized eggs derived from an intercross between Fl progeny of aC57BL/6J ob/ob X DBA intercross. These crosses require the use ofC57BL/6J ob/ob ovarian transplants to generate the Fl animals. DBA/2Jmice are used as the counterstrain because they have a nonagouti coatcolor which is important when using the ovarian transplants. Genotype atthe OB loci in cosmid transgenic animals can be determined by typinganimals with tightly linked RFLPs or microsatellites which flank themutation and which are polymorphic between the progenitor strains.Complementation will be demonstrated when a particular construct rendersa genetically obese F2 animal (as scored by RFLP analysis) lean andnondiabetic. Under these circumstances, fmal proof of complementationwill require that the oblob or db/db animal carrying the transgene bemated to the ob/ob or db/db ovarian transplants. In this cross, all N2animals which do not carry the transgene will be obese and insulinresistant/diabetic, while those that do carry the transgene will be leanand have normal glucose and insulin concentrations in plasma. In agenetic sense, the transgene acts as a suppressor mutation.

Alternatively, OB genes can be tested by examining their phenotypiceffects when expressed in antisense orientation in wild-type animals. Inthis approach, expression of the wild-type allele is suppressed, whichleads to a mutant phenotype. RNA.RNA duplex formation (antisense-sense)prevents normal handling of mRNA, resulting in partial or completeelimination of wild-type gene effect. This technique has been used toinhibit TK synthesis in tissue culture and to produce phenotypes of theKruppel mutation in Drosophila, and the Shiverer mutation in mice [Izantet al., Cell, 36:1007-1015 (1984); Green et al., Annu. Rev. Biochem.,55:569-597 (1986); Katsuki et al., Science, 241:593-595 (1988)]. Animportant advantage of this approach is that only a small portion of thegene need be expressed for effective inhibition of expression of theentire cognate mRNA. The antisense transgene will be placed undercontrol of its own promoter or another promoter expressed in the correctcell type, and placed upstream of the SV40 polyA site. This transgenewill be used to make transgenic mice. Transgenic mice will also be matedovarian transplants to test whether ob heterozygotes are more sensitiveto the effects of the antisense construct.

In the long term, the elucidation of the biochemical function of the OBgene product (the OB polypeptide or protein) is useful for identifyingsmall molecule agonists and antagonists that affect its activity.

Various terms used throughout this specification shall have thedefinitions set out herein, for example, below.

The term "body weight modulator", "modulator", "modulators", and anyvariants not specifically listed, may be used herein interchangeably,and as used throughout the present application and claims refers in oneinstance to both nucleotides and to proteinaceous material, the latterincluding both single or multiple proteins. More specifically, theaforementioned terms extend to the nucleotides and to the DNA having thesequences described herein and presented in FIG. 1A through E (SEQ IDNO:1), and FIG. 2A and B (SEQ ID NO:3). Likewise, the proteins havingthe amino acid sequence data described herein and presented in FIG. 1Athrough E (SEQ ID NO:2), and FIG. 3 (SEQ ID NO:4) are likewisecontemplated, as are the profile of activities set forth with respect toall materials both herein and in the claims. Accordingly, nucleotidesdisplaying substantially equivalent or altered activity are likewisecontemplated, including substantially homologous analogs and allelicvariations. Likewise, proteins displaying substantially equivalent oraltered activity, including proteins modified deliberately, as forexample, by site-directed mutagenesis, or accidentally through mutationsin hosts that produce the modulators are likewise contemplated.

A composition comprising "A" (where "A" is a single protein, DNAmolecule, vector, recombinant host cell, etc.) is substantially free of"B" (where "B" comprises one or more contaminating proteins, DNAmolecules, vectors, etc., but excluding racemic forms of A) when atleast about 75% by weight of the proteins, DNA, vectors (depending onthe category of species to which A and B belong) in the composition is"A". Preferably, "A" comprises at least about 90% by weight of the A+Bspecies in the composition, most preferably at least about 99% byweight. It is also preferred that a composition, which is substantiallyfree of contamination, contain only a single molecular weight specieshaving the activity or characteristic of the species of interest.

The OB Polypeptides

The terms "protein," which refers to the naturally occurringpolypeptide, and "polypeptide" are used herein interchangeably withrespect to the OB gene product and variants thereof. The term "matureprotein" or "mature polypeptide" particularly refers to the OB geneproduct with the signal sequence (or a fusion protein partner) removed.

As noted above, in specific embodiments OB polypeptides of the inventioninclude those having the amino acid sequences set forth herein e.g., SEQID NOS: 2, 4, 5, 6, etc., including the OB polypeptide modified withconservative amino acid substitutions, as well as biologically activefragments, analogs, and derivatives thereof. The term "biologicallyactive," is used herein to refer to a specific effect of thepolypeptide, including but not limited to specific binding, e.g., to areceptor, antibody, or other recognition molecule; activation of signaltransduction pathways on a molecular level; and/or induction (orinhibition by antagonists) of physiological effects mediated by thenative OB polypeptide in vivo. OB polypeptides, including fragments,analogs, and derivatives, can be prepared synthetically, e.g., using thewell known techniques of solid phase or solution phase peptidesynthesis. Preferably, solid phase synthetic techniques are employed.Alternatively, OB polypeptides of the invention can be prepared usingwell known genetic engineering techniques, as described infra. In yetanother embodiment, the OB polypeptide can be purified, e.g., byimmunoaffmity purification, from a biological fluid, such as but notlimited to plasma, serum, or urine, preferably human plasma, serum, orurine, and more preferably from a subject who overexpresses thepolypeptide, such as an obese person suffering from a mutation in the OBreceptor or from obesity related to a mutation corresponding to "fatty."

Fragments of the OB Polypeptide

In a particular embodiment, the present invention contemplates thatnaturally occurring fragments of the OB polypeptide may be important.The peptide sequence includes a number of sites that are frequently thetarget for proteolytic cleavage, e.g., arginine residues. It is possiblethat the full length polypeptide may be cleaved at one or more suchsites to form biologically active fragments. Such biologically activefragments may either agonize or antagonize the functional activity ofthe OB polypeptide to reduce body weight.

Analogs of the OB Polypeptide

The present invention specifically contemplates preparation of analogsof the OB peptide, which are characterized by being capable of abiological activity of OB polypeptide, e.g., of binding to a specificbinding partner of OB peptide, such as the OB receptor. In oneembodiment, the analog agonizes OB activity, i.e., it functionssimilarly to the OB peptide. Preferably, an OB agonist is more effectivethan the native protein. For example, an OB agonist analog may bind tothe OB receptor with higher affinity, or demonstrate a longer half-lifein vivo, or both. Nevertheless, OB peptide agonist analogs that are lesseffective than the native protein are also contemplated. In anotherembodiment, the analog antagonizes OB activity. For example, an OBanalog that binds to the OB receptor but does not induce signaltransduction can competitively inhibit binding of native OB to thereceptor, thus decreasing OB activity in vivo. Such an OB antagonistanalog may also demonstrate different properties from ob peptide, e.g.,longer (or shorter) half-life in vivo, greater (or lesser) bindingaffinity for the OB receptor, or both.

In one embodiment, an analog of OB peptide is the OB peptide modified bysubstitution of amino acids at positions on the polypeptide that are notessential for structure or function. For example, since it is known thathuman OB peptide is biologically active in mouse, substitution ofdivergent amino acid residues in the human sequence as compared to themurine amino acid sequence will likely yield useful analogs of OBpeptide. For example, the serine residue at position 53 or position 98,or both (in the unprocessed peptide sequence depicted in FIG. 4) fromhuman may be substituted, e.g., with glycine, alanine, valine, cysteine,methionine, or threonine. Similarly, the arginine residue at positionnumber 92 (FIG. 4) may be substituted, e.g., with asparagine, lysine,histidine, glutamine, glutamic acid, aspartic acid, serine, threonine,methionine, or cysteine. Referring still to FIG. 4, other amino acids inthe human OB peptide that appear to be capable of substitution arelysine at position 56, threonine at position 71, isoleucine at position85, methionine at position 89, isoleucine at position 95, valine atposition 110, histidine at position 118, tryptophan at position 121,alanine at position 122, glutamic acid at position 126, threonine atposition 127, leucine at position 128, aspartic acid at position 129,glycine at position 132, glycine at position 139, methionine at position157, tryptophan at position 159, leucine at position 163, and glycine atposition 166. In another embodiment, it may be possible to substituteone or more of residues 121 to 128 (as depicted in FIG. 4), e.g., withglycines or alanines, or substituting some of the residues with theexceptions of serine as position 123, or leucine at position 125.

In another embodiment, an analog of the OB polypeptide, preferably thehuman OB polypeptide, is a truncated form of the polypeptide. Forexample, it has already been demonstrated that the glutamine at residue49 is not essential, and can be deleted from the peptide. Similarly, itmay be possible to delete some or all of the divergent amino acidresidues at positions 121-128. In addition, the invention contemplatesproviding an OB analog having the minimum amino acid sequence necessaryfor a biological activity. This can be readily determined, e.g., bytesting the activity of fragments of OB for the ability to bind toOB-specific antibodies, inhibit the activity of the native OBpolypeptide, or agonize the activity of the native OB peptide. In oneembodiment, the invention provides a truncated OB polypeptide consistingof the loop structure formed by the disulfide bond that forms betweencysteine residues 117 and 167 (as depicted in FIG. 4). In anotherembodiment, the truncated analog corresponds to the amino acids fromresidue 22 (which follows the putative signal peptide cleavage site) to53 [the amino acid residue immediately preceding a flexible loop regiondetected with limited proteolysis followed by mass spectrometricanalysis of the 013 polypeptide; see Cohen et al., Protein Science,4:1088 (1995)]. In another embodiment, the truncated analog correspondsto amino acids from residue 61 (the residue immediately following theflexible loop region as detected with the limited proteolysis/mass spec.analysis of the OB polypeptide) to amino acid residue 116 (the residueimmediately preceding the first cysteine residue). In yet anotherembodiment, the truncated analog corresponds to amino acids from residue61 to amino acid residue 167.

Furthermore, one or more of the residues of the putative flexible loopat residues number 54 to 60 are substituted. For example, one or more ofthe residues may be substituted with lysine, glutamic acid, or cysteine(preferably lysine) for cross linking, e.g., to a polymer, sinceflexible loop structures are preferred sites for derivatization of aprotein. Alternatively, the residues at the flexible loop positions maybe substituted with amino acid residues that are more resistant toproteolysis but that retain a flexible structure, e.g., one or moreprolines. In yet another embodiment, substitutions with amino acidresidues that can be further derivatized to make them more resistant todegradation, e.g., proteolysis, is contemplated.

It will be appreciated by one of ordinary skill in the art that theforegoing fragment sizes are approximate, and that from one to aboutfive amino acids can be included or deleted from each or both ends, orfrom the interior of the polypeptide or fragments thereof, of therecited truncated analogs, with the exception that in the disulfidebonded loop analogs, the cysteine residues must be maintained.

It has been found that murine OB peptide contains 50% α-helical content,and that the human OB polypeptide contains about 60% α-helical content,as detected by circular dichroism of the recombinant peptides undernearly physiological conditions. Accordingly, in another embodiment,amino acid residues can be substituted with residues to form analogs ofOB polypeptide that demonstrate enhanced propensity for formiing, orwhich form more stable, α-helix structures. For example, α-helixstructure would be preferred if Glu, Ala, Leu, His, Trp are introducedas substitutes for amino acid residues found in the native OBpolypeptide. Preferably, conservative amino acid substitutions areemployed, e.g., substituting aspartic acid at residue(s) 29, 30, 44, 61,76, 100, and/or 106 (as depicted in FIG. 4) with glutamic acid(s) (Glu);substituting isoleucine(s) with leucine; substituting glycine or valine,or any divergent amino acid, with alanine (e.g., serine at position 53of the human OB polypeptide with alanine), substituting arginine orlysine with histidine, and substituting tyrosine and/or phenylalaninewith tryptophan. Increasing the degree, or more importantly, thestability of α-helix structure may yield an OB analog with greateractivity, increased binding affinity, or longer half-life. In a specificembodiment, the helix forming potential of the portion of the OB peptidecorresponding to amino acid residues 22 through 53 is increased. Inanother embodiment, the helix-forming potential or stability of theamino acid residues 61-116 is increased. In yet another embodiment, thehelix forming potential of the disulfide loop structure corresponding toamino acids 117 to 167 is increased. Also contemplated are OB analogscontaining enhanced α-helical potential or stability in more than one ofthe foregoing domains. In a further embodiment, truncated OB polypeptideanalogs are generated that incorporate structure-forming, e.g.,helix-forming, amino acid residues to compensate for the greaterpropensity of polypeptide fragments to lack stable structure.

Analogs, such as fragments, may be produced, for example, by pepsindigestion of weight modulator peptide material. Other analogs, such asmuteins, can be produced by standard site-directed mutagenesis of weightmodulator peptide coding sequences. Analogs exhibiting "weight modulatoractivity" such as small molecules, whether functioning as promoters orinhibitors, may be identified by known in vivo and/or in vitro assays.

Small Molecule Analogs and Peptidomimetics of OB Polypeptide

The structure of the OB polypeptide, preferably human OB polypeptide,can be analyzed by various methods known in the art. The proteinsequence can be characterized by a hydrophilicity analysis [e.g., Hoppet al., Proc. Natl. Acad. Sci. USA, 78:3824 (1981)]. A hydrophilicityprofile can be used to identify the hydrophobic and hydrophilic regionsof the OB polypeptide, which may indicate regions buried in the interiorof the folded polypeptide, and regions accessible on the exterior of thepolypeptide. In addition, secondary structural analysis [e.g., Chou etal, Biochem., 13:222 (1974)] can also be done, to identify regions of OBpolypeptide that assume specific secondary structures. Manipulation ofthe predicted or determined structure, including secondary structureprediction, can be accomplished using computer software programsavailable in the art.

By providing an abundant source of recombinant OB polypeptide, thepresent invention enables quantitative structural determination of thepolypeptide. In particular, enough material is provided for nuclearmagnetic resonance (NMR), infrared (IR), Raman, and ultraviolet (UV),especially circular dichroism (CD), spectroscopic analysis. Inparticular NMR provides very powerful structural analysis of moleculesin solution, which more closely approximates their native environment[Marion et al., Biochem. Biophys. Res. Comm., 113:967-974 (1983); Bar etal., J. Magn. Reson., 65:355-360 (1985); Kimura et al., Proc. Natl.Acad. Sci. USA, 77:1681-1685 (1980)]. Other methods of structuralanalysis can also be employed. These include but are not limited toX-ray crystallography [Engstom, Biochem. Exp. Biol., 11:7-13 (1974)].

In yet a further embodiment, an analog of OB polypeptide can be testedto determine whether it cross-reacts with an antibody specific fornative OB polypeptide, or specific fragments thereof. The degree ofcross-reactivity provides information about structural homology orsimilarity of proteins, or about the accessibility of regionscorresponding to portions of the polypeptide that were used to generatefragment-specific antibodies.

Screening for OB Analogs

Various screening techniques are known in the art for screening foranalogs of polypeptides. Various libraries of chemicals are available.Accordingly, the present invention contemplates screening suchlibraries, e.g., libraries of synthetic compounds generated over yearsof research, libraries of natural compounds, and combinatoriallibraries, as described in greater detail, infra, for analogs of OBpolypeptide. In one embodiment, the invention contemplates screeningsuch libraries for compounds that bind to anti-OB polypeptideantibodies, preferably anti-human ob polypeptide antibodies. In anotheraspect, once the OB receptor is identified (see infra), any screeningtechnique known in the art can be used to screen for OB receptoragonists or antagonists. The present invention contemplates screens forsmall molecule ligands or ligand analogs and mimics, as well as screensfor natural ligands that bind to and agonize or antagonize activate OBreceptor in vivo.

Knowledge of the primary sequence of the receptor, and the similarity ofthat sequence with proteins of known function, can provide an initialclue as to the agonists or antagonists of the protein. Identificationand screening of antagonists is further facilitated by determiningstructural features of the protein, e.g., using X-ray crystallography,neutron diffraction, nuclear magnetic resonance spectrometry, and othertechniques for structure determination. These techniques provide for therational design or identification of agonists and antagonists.

Another approach uses recombinant bacteriophage to produce largelibraries. Using the "phage method" [Scott et al., Science, 249:386-390(1990); Cwirla et al., Proc. Natl. Acad. Sci. USA, 87:6378-6382 (1990);Devlin et al., Science, 249:404-406 (1990)], very large libraries can beconstructed (10⁶ -10⁸ chemical entities). A second approach usesprimarily chemical methods, of which the Geysen method [Geysen et al.,Molecular Immunology, 23:709-715 (1986); Geysen et al., J. ImmunologicMethod, 102:259-274 (1987)] and the recent method of Fodor et al.,Science, 251:767-773 (1991) are examples. Furka et al. 14thInternational Congress of Biochemistry, Volume 5, Abstract FR:013(1988); Furka, Int. J. Peptide Protein Res., 37:487-493 (1991); Houghton(U.S. Pat. No. 4,631,211, issued December 1986); and Rutter et al. (U.S.Pat. No. 5,010,175, issued Apr. 23, 1991) describe methods to produce amixture of peptides that can be tested as agonists or antagonists.

In another aspect, synthetic libraries [Needels et al., Proc. Natl.Acad. Sci. USA, 90:10700-10704 (1993); Lam et al., International PatentPublication No. WO 92/00252, each of which is incorporated herein byreference in its entirety], and the like can be used to screen for OBreceptor ligands according to the present invention. With suchlibraries, receptor antagonists can be detected using cells that expressthe receptor without actually cloning the OB receptor.

Alternatively, assays for binding of soluble ligand to cells thatexpress recombinant forms of the OB receptor ligand binding domain canbe performed. The soluble ligands can be provided readily as recombinantor synthetic OB polypeptide.

The screening can be performed with recombinant cells that express theOB receptor, or alternatively, using purified receptor protein, e.g.,produced recombinantly, as described above. For example, the ability oflabeled, soluble or solubilized OB receptor, that includes theligand-binding portion of the molecule, to bind ligand can be used toscreen libraries, as described in the foregoing references.

Derivatives of OB Polypeptides

Generally, the present protein (herein the term "protein" is used toinclude "polypeptide," unless otherwise indicated) may be derivatized bythe attachment of one or more chemical moieties to the protein moiety.The chemically modified derivatives may be further formulated forintraarterial, intraperitoneal, intramuscular, subcutaneous,intravenous, oral, nasal, rectal, bucal, sublingual, pulmonary, topical,transdermal, or other routes of administration. Chemical modification ofbiologically active proteins has been found to provide additionaladvantages under certain circumstances, such as increasing the stabilityand circulation time of the therapeutic protein and decreasingimmunogenicity. See U.S. Pat. No. 4,179,337, Davis et al., issued Dec.18, 1979. For a review, see Abuchowski et al., "Soluble Polymer-EnzymeAdducts", in Enzymes as Drugs, pp. 367-383, Holcenberg and Roberts,eds., Wiley-Interscience, New York, N.Y., (1981). A review articledescribing protein modification and fusion proteins is Francis, Focus onGrowth Factors, 3:4-10 (1992).

Chemical Moieties For Derivatization

The chemical moieties suitable for derivatization may be selected fromamong water soluble polymers. The polymer selected should be watersoluble so that the protein to which it is attached does not precipitatein an aqueous environment, such as a physiological environment.Preferably, for therapeutic use of the end-product preparation, thepolymer will be pharmaceutically acceptable. One skilled in the art willbe able to select the desired polymer based on such considerations aswhether the polymer/protein conjugate will be used therapeutically, andif so, the desired dosage, circulation time, resistance to proteolysis,and other considerations. For the present proteins and peptides, thesemay be ascertained using the assays provided herein.

Polymer Molecules

The water soluble polymer may be selected from the group consisting of,for example, polyethylene glycol, copolymers of ethyleneglycol/propylene glycol, carboxymethylcellulose, dextran, polyvinylalcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyolsand polyvinyl alcohol. Polyethylene glycol propionaldenhyde may provideadvantages in manufacturing due to its stability in water.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 2kDa and about 100kDa (the term "about" indicating that inpreparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

Polymer/Protein Ratio

The number of polymer molecules so attached may vary, and one skilled inthe art will be able to ascertain the effect on function. One maymono-derivatize, or may provide for a di-, tri-, tetra- or somecombination of derivatization, with the same or different chemicalmoieties (e.g., polymers, such as different weights of polyethyleneglycols). The proportion of polymer molecules to protein (or peptide)molecules will vary, as will their concentrations in the reactionmixture. In general, the optimum ratio (in terms of efficiency ofreaction in that there is no excess unreacted protein or polymer) willbe determined by factors such as the desired degree of derivatization(e.g., mono, di-, tri-, etc.), the molecular weight of the polymerselected, whether the polymer is branched or unbranched, and thereaction conditions.

Attachment of the Chemical Moiety to the Protein

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art, e.g., EP 0 401 384 hereinincorporated by reference (coupling PEG to G-CSF). See also Malik etal., Exp. Hematol., 20:1028-1035 (1992) (reporting pegylation of G-CSFusing tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas a free amino or carboxyl group. Reactive groups are those to which anactivated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues, those having a free carboxyl group mayinclude aspartic acid residues, glutamic acid residues and theC-terminal amino acid residue. Sulfhydryl groups may also be used as areactive group for attaching the polyethylene glycol molecule(s).Preferred for therapeutic purposes is attachment at an amino group, suchas attachment at the N-terminus or lysine group. Attachment at residuesimportant for receptor binding should be avoided if receptor binding isdesired.

N-terminally Chemically Modified Proteins

One may specifically desire N-terminally chemically modified protein.Using polyethylene glycol as an illustration of the presentcompositions, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (or peptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective N-terminal chemicalmodification may be accomplished by reductive alkylation which exploitsdifferential reactivity of different types of primary amino groups(lysine versus the N-terminus) available for derivatization in aparticular protein. Under the appropriate reaction conditions,substantially selective derivatization of the protein at the N-terminuswith a carbonyl group containing polymer is achieved. For example, onemay selectively N-terminally pegylate the protein by performing thereaction at a pH which allows one to take advantage of the pK_(a)differences between the ε-amino groups of the lysine residues and thatof the α-amino group of the N-terninal residue of the protein. By suchselective derivatization attachment of a water soluble polymer to aprotein is controlled: the conjugation with the polymer takes placepredominantly at the N-terminus of the protein and no significantmodification of other reactive groups, such as the lysine side chainamino groups, occurs. Using reductive alkylation, the water solublepolymer may be of the type described above, and should have a singlereactive aldehyde for coupling to the protein. Polyethylene glycolpropionaldehyde, containing a single reactive aldehyde, may be used.

Nucleic Acids Associated With OB Polypeptide

As noted above, the present invention is directed to nucleic acidsencoding ob polypeptides, as well as associated genomic non-codingsequences 5', 3', and intronic to the OB gene. Thus, in accordance withthe present invention there may be employed conventional molecularbiology, microbiology, and recombinant DNA techniques within the skillof the art. Such techniques are explained fully in the literature. See,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989); Glover ed., DNA Cloning: A Practical Approach, Volumes I and II,MRL Press, Ltd., Oxford, U.K. (1985); Gait ed., OligonucleotideSynthesis, Oxford University Press (1984); Hames et al., eds., NucleicAcid Hybridization, Springer-Verlag (1985); Hames et al., eds.Transcription And Translation, Oxford University Press (1984); Freshneyed., Animal Cell Culture, Oxford University Press (1986); ImmobilizedCells And Enzymes, IRL Press (1986); Perbal, A Practical Guide ToMolecular Cloning, Wiley, N.Y. (1984). Of particular relevance to thepresent invention are strategies for isolating, cloning, sequencing,analyzing, and characterizing a gene or nucleic acid based on the wellknown polymerase chain reaction (PCR) techniques.

A "replicon" is any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo, i.e.,capable of replication under its own control.

A "vector" is a replicon, such as a plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment.

A "cassette" refers to a segment of DNA that can be inserted into avector at specific restriction sites. The segment of DNA encodes apolypeptide of interest, and the cassette and restriction sites aredesigned to ensure insertion of the cassette in the proper reading framefor transcription and translation.

"Heterologous" DNA refers to DNA not naturally located in the cell, orin a chromosomal site of the cell. Preferably, the heterologous DNAincludes a gene foreign to the cell.

A cell has been "transfected" by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. A cell has been "transformed"by exogenous or heterologous DNA when the transfected DNA effects aphenotypic change. Preferably, the transforming DNA should be integrated(covalently linked) into chromosomal DNA making up the genome of thecell.

A "clone" is a population of cells derived from a single cell or commonancestor by mitosis.

A "nucleic acid molecule" refers to the phosphate ester polymeric formof ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNAmolecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine,deoxythymidine, or deoxycytidine; "DNA molecules") in eithersingle-stranded form, or a double-stranded helix. Double-strandedDNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acidmolecule, and in particular DNA or RNA molecule, refers only to theprimary and secondary structure of the molecule, and does not limit itto any particular tertiary or quaternary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear or circular DNAmolecules (e.g., restriction fragments), plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5' to 3' direction along thenontranscribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA). A "recombinant DNA molecule" is a DNA moleculethat has undergone a molecular biological manipulation.

A nucleic acid molecule is "hybridizable" to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single-strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al., 1989, supra). The conditions oftemperature and ionic strength determine the "stringency" of thehybridization. For preliminary screening for homologous nucleic acids,low stringency hybridization conditions, corresponding to a T_(m) of 55°C., can be used, e.g., 5x SSC, 0.1% SDS, 0.25% milk, and no formamide;or 30% formamide, 5x SSC, 0.5% SDS). Moderate stringency hybridizationconditions correspond to a higher T_(m), e.g., 40% formamide, with 5x or6x SCC. High stringency hybridization conditions correspond to thehighest T_(m), e.g., 50% formamide, 5x or 6x SCC. Hybridization requiresthat the two nucleic acids contain complementary sequences, althoughdepending on the stringency of the hybridization, mismatches betweenbases are possible. The appropriate stringency for hybridizing nucleicacids depends on the length of the nucleic acids and the degree ofcomplementation, variables well known in the art. The greater the degreeof similarity or homology between two nucleotide sequences, the greaterthe value of T_(m) for hybrids of nucleic acids having those sequences.The relative stability (corresponding to higher T_(m)) of nucleic acidhybridizations decreases in the following order: RNA:RNA, DNA:RNA,DNA:DNA. For hybrids of greater than 100 nucleotides in length,equations for calculating T_(m) have been derived (see Sambrook et al.,1989, supra, 9.50-0.51). For hybridization with shorter nucleic acids,i.e., oligonucleotides, the position of mismatches becomes moreimportant, and the length of the oligonucleotide determines itsspecificity (see Sambrook et al., 1989, supra, 11.7-11.8). Preferably aminimum length for a hybridizable nucleic acid is at least about 10nucleotides; more preferably at least about 15 nucleotides; mostpreferably the length is at least about 20 nucleotides.

"Homologous recombination" refers to the insertion of a foreign DNAsequence of a vector in a chromosome. Preferably, the vector targets aspecific chromosomal site for homologous recombination. For specifichomologous recombination, the vector will contain sufficiently longregions of homology to sequences of the chromosome to allowcomplementary binding and incorporation of the vector into thechromosome. Longer regions of homology, and greater degrees of sequencesimilarity, may increase the efficiency of homologous recombination.

A DNA "coding sequence" is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in a cell in vitro or invivo when placed under the control of appropriate regulatory sequences.The boundaries of the coding sequence are determined by a start codon atthe 5' (amino) terminus and a translation stop codon at the 3'(carboxyl) terminus. A coding sequence can include, but is not limitedto, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNAsequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNAsequences. If the coding sequence is intended for expression in aeukaryotic cell, a polyadenylation signal and transcription terminationsequence will usually be located 3' to the coding sequence.

Isolation of OB Coding and Flanking Sequences

The nucleic acids contemplated by the present invention extend asindicated, to other nucleic acids that code on expression for peptidessuch as those set forth in FIG. 1A through D (SEQ ID NO:2), FIG. 3 (SEQID NO:4), FIG. 5 (SEQ ID NO:5), and FIG. 6 (SEQ ID NO:6) herein.Accordingly, while specific DNA has been isolated and sequenced inrelation to the ob gene, any animal cell potentially can serve as thenucleic acid source for the molecular cloning of a gene encoding thepeptides of the invention. The DNA may be obtained by standardprocedures known in the art from cloned DNA (e.g., a DNA "library"), bychemical synthesis, by cDNA cloning, or by the cloning of genomic DNA,or fragments thereof, purified from the desired cell (See, for example,Sambrook et aL, 1989, supra; Glover, 1985, supra). Clones derived fromgenomic DNA may contain regulatory and intronic DNA regions in additionto coding regions; clones derived from cDNA will not contain intronsequences. Whatever the source, the gene should be molecularly clonedinto a suitable vector for propagation of the gene.

In the molecular cloning of the gene from genomic DNA, the genomic DNAcan be amplified using primers selected from the cDNA sequences.Alternatively, DNA fragments are generated, some of which will encodethe desired gene. The DNA may be cleaved at specific sites using variousrestriction enzymes. One may also use DNase in the presence of manganeseto fragment the DNA, or the DNA can be physically sheared, as forexample, by sonication. The linear DNA fragments can then be separatedaccording to size by standard techniques, including but not limited to,agarose and polyacrylamide gel electrophoresis and columnchromatography.

Once the DNA fragments are generated, identification of the specific DNAfragment containing the desired OB or OB-like gene may be accomplishedin a number of ways. For example, if an amount of a portion of a OB orOB-like gene or its specific RNA, or a fragment thereof, is availableand can be purified and labeled, the generated DNA fragments may bescreened by nucleic acid hybridization to a labeled probe [Benton etal., Science, 196:180 (1977); Grunstein et al., Proc. Natl. Acad. Sci.USA, 72:3961 (1975)]. The present invention provides such nucleic acidprobes, which can be conveniently prepared from the specific sequencesdisclosed herein, e.g., a hybridizable probe having a nucleotidesequence corresponding to at least a 10, and preferably a 15, nucleotidefragment of the sequences depicted in FIG. 1A through E (SEQ ID NO:1) orFIG. 2A and B (SEQ ID NO:3). Preferably, a fragment is selected that ishighly unique to the modulator peptides of the invention. Those DNAfragments with substantial homology to the probe will hybridize. Asnoted above, the greater the degree of homology, the more stringent thehybridization conditions that can be used. In one embodiment, lowstringency hybridization conditions are used to identify a homologousmodulator peptide. However, in a preferred aspect, and as demonstratedexperimentally herein, a nucleic acid encoding a modulator peptide ofthe invention will hybridize to a nucleic acid having a nucleotidesequence such as depicted in FIG. 1A through E (SEQ ID NO:1) or FIG. 2Aand B (SEQ ID NO:3), or a hybridizable fragment thereof, undermoderately stringent conditions; more preferably, it will hybridizeunder high stringency conditions.

Alternatively, the presence of the gene may be detected by assays basedon the physical, chemical, or immunological properties of its expressedproduct. For example, cDNA clones, or DNA clones which hybrid-select theproper mRNAs, can be selected which produce a protein that, e.g., hassimilar or identical electrophoretic migration, isoelectric focusingbehavior, proteolytic digestion maps, tyrosine phosphatase activity orantigenic properties as known for the present modulator peptides. Forexample, the antibodies of the instant invention can conveniently beused to screen for homologs of modulator peptides from other sources.

A gene encoding a modulator peptide of the invention can also beidentified by mRNA selection, i.e., by nucleic acid hybridizationfollowed by in vitro translation. In this procedure, fragments are usedto isolate complementary mRNAs by hybridization. Such DNA fragments mayrepresent available, purified modulator DNA. Immunoprecipitationanalysis or functional assays (e.g., tyrosine phosphatase activity) ofthe in vitro translation products of the products of the isolated mRNAsidentifies the mRNA and, therefore, the complementary DNA fragments,that contain the desired sequences. In addition, specific mRNAs may beselected by adsorption of polysomes isolated from cells to immobilizedantibodies specifically directed against a modulator peptide.

A radiolabeled modulator peptide cDNA can be synthesized using theselected mRNA (from the adsorbed polysomes) as a template. Theradiolabeled mRNA or cDNA may then be used as a probe to identifyhomologous modulator peptide DNA fragments from among other genomic DNAfragments.

As mentioned above, a DNA sequence encoding weight modulator peptides asdisclosed herein can be prepared synthetically rather than cloned. TheDNA sequence can be designed with the appropriate codons for the weightmodulator peptide amino acid sequences. In general, one will selectpreferred codons for the intended host if the sequence will be used forexpression. The complete sequence is assembled from overlappingoligonucleotides prepared by standard methods and assembled into acomplete coding sequence. See, e.g., Edge, Nature, 292:756 (1981);Nambair et al., Science, 223:1299 (1984); Jay et al., J. Biol. Chem.,259:6311 (1984).

Synthetic DNA sequences allow convenient construction of genes whichwill express weight modulator analogs, as described above.Alternatively, DNA encoding analogs can be made by site-directedmutagenesis of native OB genes or cDNAs, and analogs can be madedirectly using conventional polypeptide synthesis.

A general method for site-specific incorporation of unnatural aminoacids into proteins is described in Noren et al, Science, 244:182-188(1989). This method may be used to create analogs of the OB polypeptidewith unnatural amino acids.

Non-coding Nucleic Acids

The present invention extends to the preparation of antisensenucleotides and ribozymes that may be used to interfere with theexpression of the weight modulator proteins at the translational level.This approach utilizes antisense nucleic acid and ribozymes to blocktranslation of a specific mRNA, either by masking that mRNA with anantisense nucleic acid or cleaving it with a ribozyme.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule [See, Weintraub, Sci.Am., 262:40-46 (1990); Marcus-Sekura, Anal. Biochem., 172:289-295(1988)]. In the cell, they hybridize to that mRNA, forming adouble-stranded molecule. The cell does not translate an mRNA complexedin this double-stranded form. Therefore, antisense nucleic acidsinterfere with the expression of mRNA into protein. Oligomers of aboutfifteen nucleotides and molecules that hybridize to the AUG initiationcodon will be particularly efficient, since they are easy to synthesizeand are likely to pose fewer problems than larger molecules whenintroducing them into weight modulator peptide-producing cells.Antisense methods have been used to inhibit the expression of many genesin vitro [(Marcus-Sekura, 1988 supra; Hambor et al., J. Exp. Med.,168:1237-1245 (1988)].

Ribozymes are RNA molecules possessing the ability to specificallycleave other single- stranded RNA molecules in a manner somewhatanalogous to DNA restriction endonucleases. Ribozymes were discoveredfrom the observation that certain mRNAs have the ability to excise theirown introns. By modifying the nucleotide sequence of these RNAs,researchers have been able to engineer molecules that recognize specificnucleotide sequences in an RNA molecule and cleave it [Cech, J. Am. Med.Assoc., 260:3030-3034 (1988)]. Because they are sequence-specific, onlymRNAs with particular sequences are inactivated.

Investigators have identified two types of ribozymes, Tetrahymena-typeand "hammerhead"-type. Tetrahymena-type ribozymes recognize four-basesequences, while "hammerhead"-type recognize eleven- to eighteen-basesequences. The longer the recognition sequence, the more likely it is tooccur exclusively in the target mRNA species. Therefore, hammerhead-typeribozymes are preferable to Tetrahymena-type ribozymes for inactivatinga specific mRNA species, and eighteen base recognition sequences arepreferable to shorter recognition sequences.

The DNA sequences described herein may thus be used to prepare antisensemolecules against and ribozymes that cleave mRNAs for weight modulatorproteins and their ligands, thus inhibiting expression of the OB gene,and leading to increased weight gain and adiposity.

In another embodiment, short oligonucleotides complementary to thecoding and complementary strands of the OB nucleic acid, or tonon-coding regions of the OB gene 5', 3', or internal (intronic) to thecoding region are provided by the present invention. Such nucleic acidsare useful as probes, either as directly labeled oligonucleotide probes,or as primers for the polymerase chain reaction, for evaluating thepresence of mutations in the OB gene, or the level of expression of OBmRNA. Preferably, the non-coding nucleic acids of the invention are fromthe human OB gene.

In a specific embodiment, the non-coding nucleic acids provide forhomologous recombination for integration of an amplifiable gene and/orother regulatory sequences in proximity to the OB gene, e.g., to providefor higher levels of expression of the OB polypeptide, or to overcome amutation in the OB gene regulatory sequences that prevent proper levelsof expression of the OB polypeptide (See International PatentPublication WO 91/06666, published May 16, 1991 by Skoultchi;International Patent Publication No. WO 91/09955, published Jul. 11,1991 by Chappel; see also International Patent Publication No. WO90/14092, published Nov. 29, 1990, by Kucherlapati and Campbell).

Production of OB Polypeptide: Expression and Synthesis

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, terminators, and the like, thatprovide for the expression of a coding sequence in a host cell. Ineukaryotic cells, polyadenylation signals are control sequences.

A coding sequence is "under the control" of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then trans-RNAspliced and translated into the protein encoded by the coding sequence.

A "signal sequence" is included at the beginning of the coding sequenceof a protein to be expressed on the surface of a cell. This sequenceencodes a signal peptide, N-terminal to the mature polypeptide, thatdirects the host cell to translocate the polypeptide. The term"translocation signal sequence" is also used herein to refer to thissort of signal sequence. Translocation signal sequences can be foundassociated with a variety of proteins native to eukaryotes andprokaryotes, and are often functional in both types of organisms.

A DNA sequence is "operatively linked" to an expression control sequencewhen the expression control sequence controls and regulates thetranscription and translation of that DNA sequence. The term"operatively linked" includes having an appropriate start signal (e.g.,ATG) in front of the DNA sequence to be expressed and maintaining thecorrect reading frame to permit expression of the DNA sequence under thecontrol of the expression control sequence and production of the desiredproduct encoded by the DNA sequence. If a gene that one desires toinsert into a recombinant DNA molecule does not contain an appropriatestart signal, such a start signal can be inserted upstream (5') of andin reading frame with the gene.

A "promoter sequence" is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3'direction) coding sequence. For purposes of defimnig the presentinvention, the promoter sequence is bounded at its 3' terminus by thetranscription initiation site and extends upstream (5' direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

Another feature of this invention is the expression of the DNA sequencesdisclosed herein. As is well known in the art, DNA sequences may beexpressed by operatively linking them to an expression control sequencein an appropriate expression vector and employing that expression vectorto transform an appropriate unicellular host.

Such operative linking of a DNA sequence of this invention to anexpression control sequence, of course, includes, if not already part ofthe DNA sequence, the provision of an initiation codon, ATG, in thecorrect reading frame upstream of the DNA sequence.

A wide variety of host/expression vector combinations may be employed inexpressing the DNA sequences of this invention. Useful expressionvectors, for example, may consist of segments of chromosomal,non-chromosomal and synthetic DNA sequences. Suitable vectors includederivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmidscol El, pCR1, pBR322, pMB9, pUC or pUC plasmid derivatives, e.g., pGEXvectors, pET vectors, pmal-c, pFLAG, etc., and their derivatives,plasmids such as RP4; phage DNAs, e.g., the numerous derivatives ofphage λ, e.g., NM989, and other phage DNA, e.g., M13 and filamentoussingle-stranded phage DNA; yeast plasmids such as the 2μ plasmid orderivatives thereof; vectors useful in eukaryotic cells, such as vectorsuseful in insect or mammalian cells; vectors derived from combinationsof plasmids and phage DNAs, such as plasmids that have been modified toemploy phage DNA or other expression control sequences; and the like. Ina preferred embodiment, expression of OB is achieved in methylotrophicyeast, e.g., Pichia pastoris yeast (see, e.g., International PatentPublication No. WO 90/03431, published Apr. 5, 1990, by Brierley et al.;International Patent Publication No. WO 90/10697, published Sep. 20,1990, by Siegel et al.). In a specific embodiment, infra, an expressionvector is engineered for expression of OB under control of the α-matingfactor signal sequence.

Any of a wide variety of expression control sequences--sequences thatcontrol the expression of a DNA sequence operatively linked to it--maybe used in these vectors to express the DNA sequences of this invention.Such useful expression control sequences include, for example, the earlyor late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lacsystem, the trp system, the TAC system, the TRC system, the LTR system,the major operator and promoter regions of phage λ, the control regionsof fd coat protein, the promoter for 3-phosphoglycerate kinase or otherglycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), theAOX 1 promoter of methylotrophic yeast, the promoters of the yeastα-mating factors, and other sequences known to control the expression ofgenes of prokaryotic or eukaryotic cells or their viruses, and variouscombinations thereof.

A wide variety of unicellular host cells are also useful in expressingthe DNA sequences of this invention. These hosts may include well knowneukaryotic and prokaryotic hosts, such as strains of E. coli,Pseudomonas, Bacillus, Streptomyces; fungi such as yeasts(Saccharomyces, and methylotrophic yeast such as Pichia, Candida,Hansenula, and Torulopsis); and animal cells, such as CHO, Rl.1, B-W andLM cells, African Green Monkey kidney cells (e.g., COS 1, COS 7, BSC1,BSC40, and BMT10), insect cells (e.g., Sf9), and human cells and plantcells in tissue culture.

It will be understood that not all vectors, expression control sequencesand hosts will function equally well to express the DNA sequences ofthis invention. Neither will all hosts function equally well with thesame expression system. However, one skilled in the art will be able toselect the proper vectors, expression control sequences, and hostswithout undue experimentation to accomplish the desired expressionwithout departing from the scope of this invention. For example, inselecting a vector, the host must be considered because the vector mustfunction in it. The vector's copy number, the ability to control thatcopy number, and the expression of any other proteins encoded by thevector, such as antibiotic markers, will also be considered.

In selecting an expression control sequence, a variety of factors willnormally be considered. These include, for example, the relativestrength of the system, its controllability, and its compatibility withthe particular DNA sequence or gene to be expressed, particularly asregards potential secondary structures. Suitable unicellular hosts willbe selected by consideration of, e.g., their compatibility with thechosen vector, their secretion characteristics, their ability to foldproteins correctly, and their fermentation requirements, as well as thetoxicity to the host of the product encoded by the DNA sequences to beexpressed, and the ease of purification of the expression products.

Considering these and other factors, a person skilled in the art will beable to construct a variety of vector/expression control sequence/hostcombinations that will express the DNA sequences of this invention onfermentation or in large scale animal culture.

In a specific embodiment, an OB fusion protein can be expressed. An OBfusion protein comprises at least a functionally active portion of anon-OB protein joined via a peptide bond to at least a functionallyactive portion of an OB polypeptide. The non-OB sequences can be amino-or carboxy-terminal to the OB sequences. More preferably, for stableexpression of a proteolytically inactive OB fusion protein, the portionof the non-OB fusion protein is joined via a peptide bond to theamino-terminus of the OB protein. A recombinant DNA molecule encodingsuch a fusion protein comprises a sequence encoding at least afunctionally active portion of a non-OB protein joined in-frame to theOB coding sequence, and preferably encodes a cleavage site for aspecific protease, e.g., thrombin or Factor Xa, preferably at theOB-non-OB juncture. In a specific embodiment, the fusion protein isexpressed in Escherichia coli or in P. pastoris.

In a specific embodiment, infra, vectors were prepared to express themurine and human OB genes, with and without the codon for gln-49, inbacterial expression systems and yeast (Pichia) expression systems asfusion proteins. The OB gene is prepared with an endonuclease cleavagesite, e.g., using PCR and novel primers. It is desirable to confirmsequences generated by PCR, since the probability of including a pointmutation is greater with this technique. A plasmid containing ahistidine tag (His-tag) and a proteolytic cleavage site is used. Thepresence of the histidine makes possible the selective isolation ofrecombinant proteins on a Ni-chelation column, or by affinitypurification. The proteolytic cleavage site, in a specific embodiment,infra, a thrombin cleavage site, is engineered so that treatment withthe protease, e.g., thrombin, will release the full-length mature (i.e.,lacking a signal sequence) OB polypeptide.

In another aspect, the pGEX vector [Smith et al., Gene, 67:31-40 (1988)]can be used. This vector fuses the schistosoma japonicum glutathionineS-transferase cDNA to the sequence of interest. Bacterial proteins areharvested and recombinant proteins can be quickly purified on a reducedglutathione affinity column. The GST carrier can subsequently be cleavedfrom fusion proteins by digestion with site-specific proteases. Aftercleavage, the carrier and uncleaved fusion protein can be removed byabsorption on glutathione agarose. Difficulty with the systemoccasionally arises when the encoded protein is insoluble in aqueoussolutions.

Expression of recombinant proteins in bacterial systems may result inincorrect folding of the expressed protein, requiring refolding. Therecombinant protein can be refolded prior to or after cleavage to form afunctionally active OB polypeptide. The OB polypeptide may be refoldedby the steps of (i) incubating the protein in a denaturing buffer thatcontains a reducing agent, and then (ii) incubating the protein in abuffer that contains an oxidizing agent, and preferably also contains aprotein stabilizing agent or a chaotropic agent, or both. Suitable redox(reducing/oxidizing) agent pairs include, but are not limited to,reduced glutathione/glutathione disulfide, cystine/cysteine,cystamine/cysteamine, and 2-mercaptoethanol/2-hydroxyethyldisulfide. Ina particular aspect, the fusion protein can be solubilized in adenaturant, such as urea, prior to exchange into the reducing buffer. Inpreferred embodiment, the protein is also purified, e.g., by ionexchange or Ni-chelation chromatography, prior to exchange into thereducing buffer. Denaturing agents include but are not limited to ureaand guanidine-HCl. The recombinant protein is then diluted about atleast 10-fold, more preferably about 100-fold, into an oxidizing bufferthat contains an oxidizing agent, such as but not limited to 0.1 MTris-HCl, pH 8.0, 1 mM EDTA, 0.15 M NaCl, 0.3 M oxidized glutathione.The fusion protein is then incubated for about 1 to about 24 hours,preferably about 2 to about 16 hours, at room temperature in theoxidizing buffer. The oxidizing buffer may comprise a proteinstabilizing agent, e.g., a sugar, an alcohol, or ammonium sulfate. Theoxidizing buffer may further comprises a chaotropic agent at lowconcentration, to destabilize incorrect intermolecular interactions andthus promote proper folding. Suitable chaotropic agents include but arenot limited to a detergent, a polyol, L-arginine, guanidine-HCl andpolyethylene glycol (PEG). It is important to use a low enoughconcentration of the chaotropic agent to avoid denaturing the protein.The refolded protein can be concentrated by at least about 10-fold, morepreferably by the amount it was diluted into the oxidizing buffer.

Bacterial fermentation processes can also result in a recombinantprotein preparation that contains unacceptable levels of endotoxins.Therefore, the invention contemplates removal of such endotoxins, e.g.,by using endotoxin-specific antibodies or other endotoxin bindingmolecules. The presence of endotoxins can be determined by standardtechniques, such as by employing E-TOXATE Reagents (Sigma, St. Louis,Mo.), or with bioassays.

In addition to the specific example, the present inventors contemplateuse of baculovirus, mammalian, and yeast expression systems to expressthe ob protein. For example, in baculovirus expression systems, bothnon-fusion transfer vectors, such as but not limited to pVL941 (BamH1cloning site; Summers), pVL1393 (BamH1, SmaI, XbaI, EcoR1, NotI, XmaIII,BglII, and PstI cloning site; Invitrogen), pVL1392 (BglII, PstI, NotI,XmaIII, EcoRI, XbaI, SmaI, and BamH1 cloning site; Summers andInvitrogen), and pBlueBacIII (BamH1, BglII, PstI, NcoI, and HindlIIcloning site, with blue/white recombinant screening possible;Invitrogen), and fusion transfer vectors, such as but not limited topAc700 (BamH1 and KpnI cloning site, in which the BamH1 recognition sitebegins with the initiation codon; Summers), pAc701 and pAc702 (same aspAc700, with different reading frames), pAc360 (BamH1 cloning site 36base pairs downstream of a polyhedrin initiation codon;Invitrogen(195)), and pBlueBacHisA, B, C (three different readingframes, with BamH1, BglII, PstI, NcoI, and HindIII cloning site, anN-terminal peptide for ProBond purification, and blue/white recombinantscreening of plaques; Invitrogen (220)).

Mammalian expression vectors contemplated for use in the inventioninclude vectors with inducible promoters, such as the dihydrofolatereductase (DHFR) promoter, e.g., any expression vector with a DHFRexpression vector, or a DHFR/methotrexate co-amplification vector, suchas pED (PstI, SalI, SbaI, SmaI, and EcoRI cloning site, with the vectorexpressing both the cloned gene and DHFR; see Kaufman, Current Protocolsin Molecular Biology, 16:12 (1991). Alternatively, a glutaminesynthetase/methionine sulfoximine co-amplification vector, such as pEE14(HindIII, XbaI, Smal, SbaI, EcoRI, and BclI cloning site, in which thevector expresses glutamine synthase and the cloned gene; Celltech). Inanother embodiment, a vector that directs episomal expression undercontrol of Epstein Barr Virus (EBV) can be used, such as pREP4 (BamH1,SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII, and KpnI cloning site,constitutive RSV-LTR promoter, hygromycin selectable marker;Invitrogen), pCEP4 (BamH1, SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII,and KpnI cloning site, constitutive hCMV immediate early gene,hygromycin selectable marker; Invitrogen), pMEP4 (KpnI, Pvul, NheI,HindIII, NotI, XhoI, SfiI, BamH1 cloning site, induciblemethallothionein IIa gene promoter, hygromycin selectable marker:Invitrogen), pREP8 (BamH1, XhoI, NotI, HindIII, NheI, and KpnI cloningsite, RSV-LTR promoter, histidinol selectable marker; Invitrogen), pREP9(KpnI, NheI, HindIII, NotI, XhoI, SfiI, and BamHI cloning site, RSV-LTRpromoter, G418 selectable marker; Invitrogen), and pEBVHis (RSV-LTRpromoter, hygromycin selectable marker, N-terminal peptide purifiablevia ProBond resin and cleaved by enterokinase; Invitrogen). Selectablemammalian expression vectors for use in the invention include pRc/CMV(HindlIl, BstXI, NotI, SbaI, and ApaI cloning site, G418 selection;Invitrogen), pRc/RSV (HindIII, SpeI, BstXI, NotI, XbaI cloning site,G418 selection; Invitrogen), and others. Vaccinia virus mammalianexpression vectors (see, Kaufman, 1991, supra) for use according to theinvention include but are not limited to pSC11 (SmaI cloning site, TK-and β-gal selection), pMJ601 (SalI, SmaI, AflI, NarI, BspMII, BamHI,Apal, NheI, SacII, KpnI, and HindIII cloning site; TK- and β-galselection), and pTKgptF1S (EcoRI, PstI, SalI, AccI, HindII, SbaI, BamHI,and Hpa cloning site, TK or XPRT selection).

Yeast expression systems can also be used according to the invention toexpress OB polypeptide. For example, the non-fusion pYES2 vector (XbaI,SphI, Shol, NotI, GstXI, EcoRI, BstXI, BamH1, SacI, Kpn1, and HindIlIcloning sit; Invitrogen) or the fusion pYESHisA, B, C (XbaI, SphI, ShoI,NotI, BstXI, EcoRI, BamH1, SacI, KpnI, and HindIII cloning site,N-terninal peptide purified with ProBond resin and cleaved withenterokinase; Invitrogen), to mention just two, can be employedaccording to the invention.

It is further intended that body weight modulator peptide analogs may beprepared from nucleotide sequences derived within the scope of thepresent invention.

In addition to recombinant expression of OB polypeptide, the presentinvention envisions and fully enables preparation of OB polypeptide, orfragments thereof, using the well known and highly developed techniquesof solid phase peptide synthesis. The invention contemplates using boththe popular Boc and Fmoc, as well as other protecting group strategies,for preparing OB polypeptide or fragments thereof. Various techniquesfor refolding and oxidizing the cysteine side chains to form a disulfidebond are also well-known in the art.

Antibodies to the OB Polypeptide

According to the invention, OB polypeptide produced recombinantly or bychemical synthesis, and fragments or other derivatives or analogsthereof, including fusion proteins, may be used as an immunogen togenerate antibodies that recognize the OB polypeptide. Such antibodiesinclude but are not limited to polyclonal, monoclonal, chimeric, singlechain, Fab fragments, and an Fab expression library.

A molecule is "antigenic" when it is capable of specifically interactingwith an antigen recognition molecule of the immune system, such as animmunoglobulin (antibody) or T cell antigen receptor. An antigenicpolypeptide contains at least about 5, and preferably at least about 10,amino acids. An antigenic portion of a molecule can be that portion thatis immunodominant for antibody or T cell receptor recognition, or it canbe a portion used to generate an antibody to the molecule by conjugatingthe antigenic portion to a carrier molecule for immunization. A moleculethat is antigenic need not be itself immunogenic, i.e., capable ofeliciting an immune response without a carrier.

An "antibody" is any immunoglobulin, including antibodies and fragmentsthereof, that binds a specific epitope. The term encompasses polyclonal,monoclonal, and chimeric antibodies, the last mentioned described infurther detail in U.S. Pat. Nos. 4,816,397 and 4,816,567, as well asantigen binding portions of antibodies, including Fab, F(ab')₂ and F(v)(including single chain antibodies). Accordingly, the phrase "antibodymolecule" in its various grammatical forms as used herein contemplatesboth an intact immunoglobulin molecule and an immunologically activeportion of an immunoglobulin molecule containing the antibody combiningsite. An "antibody combining site" is that structural portion of anantibody molecule comprised of heavy and light chain variable andhypervariable regions that specifically binds antigen.

Exemplary antibody molecules are intact immunoglobulin molecules,substantially intact immunoglobulin molecules and those portions of animmunoglobulin molecule that contains the paratope, including thoseportions known in the art as Fab, Fab', F(ab')₂ and F(v), which portionsare preferred for use in the therapeutic methods described herein.

Fab and F(ab')₂ portions of antibody molecules are prepared by theproteolytic reaction of papain and pepsin, respectively, onsubstantially intact antibody molecules by methods that are well-known.See for example, U.S. Pat. No. 4,342,566 to Theofilopolous et al. Fab'antibody molecule portions are also well-known and are produced fromF(ab')₂ portions followed by reduction of the disulfide bonds linkingthe two heavy chain portions as with mercaptoethanol, and followed byalkylation of the resulting protein mercaptan with a reagent such asiodoacetamide. An antibody containing intact antibody molecules ispreferred herein.

The phrase "monoclonal antibody" in its various grammatical forms refersto an antibody having only one species of antibody combining sitecapable of immunoreacting with a particular antigen. A monoclonalantibody thus typically displays a single binding affinty for anyantigen with which it immunoreacts. A monoclonal antibody may thereforecontain an antibody molecule having a plurality of antibody combiningsites, each immunospecific for a different antigen; e.g., a bispecific(chimeric) monoclonal antibody.

The term "adjuvant" refers to a compound or mixture that enhances theimmune response to an antigen. An adjuvant can serve as a tissue depotthat slowly releases the antigen and also as a lymphoid system activatorthat non-specifically enhances the immune response [Hood et al., inImmunology, p. 384, Second Ed., Benjamin/Cummings, Menlo Park, Calif.(1984)]. Often, a primary challenge with an antigen alone, in theabsence of an adjuvant, will fail to elicit a humoral or cellular immuneresponse. Adjuvants include, but are not limited to, complete Freund'sadjuvant, incomplete Freund's adjuvant, saponin, mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum. Preferably, the adjuvant is pharmaceutically acceptable.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to OB polypeptide, or fragment, derivative oranalog thereof. For the production of antibody, various host animals canbe immunized by injection with the OB polypeptide, or a derivative(e.g., fragment or fusion protein) thereof, including but not limited torabbits, mice, rats, sheep, goats, etc. In one embodiment, the OBpolypeptide or fragment thereof can be conjugated to an immunogeniccarrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin(KLH). Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

For preparation of monoclonal antibodies directed toward the OBpolypeptide, or fragment, analog, or derivative thereof, any techniquethat provides for the production of antibody molecules by continuouscell lines in culture may be used. These include but are not limited tothe hybridoma technique originally developed by Kohler et al., Nature,256:495-497 (1975), as well as the trioma technique, the human B-cellhybridoma technique [Kozbor et al., Immunology Today, 4:72 (1983)], andthe EBV-hybridoma technique to produce human monoclonal antibodies [Coleet al., in Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R.Liss, Inc., (1985)]. Immortal, antibody-producing cell lines can becreated by techniques other than fusion, such as direct transformationof B lymphocytes with oncogenic DNA, or transfection with Epstein-Barrvirus. See, e.g., M. Schreier et aL, "Hybridoma Techniques" (1980);Hammerling et al., "Monoclonal Antibodies And T-cell Hybridomas" (1981);Kennett et al., "Monoclonal Antibodies" (1980); see also U.S. Pat. Nos.4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917;4,472,500; 4,491,632; and 4,493,890.

In an additional embodiment of the invention, monoclonal antibodies canbe produced in germ-free animals utilizing recent technology(PCT/US90/02545). According to the invention, human antibodies may beused and can be obtained by using human hybridomas [Cote et al., Proc.Natl. Acad. Sci. USA, 80:2026-2030 (1983)] or by transforming human Bcells with EBV virus in vitro (Cole et al., 1985, supra). In fact,according to the invention, techniques developed for the production of"chimeric antibodies" [Morrison et al., J. Bacteriol., 159-870 (1984);Neuberger et al., Nature, 312:604-608 (1984); Takeda et al., Nature,314:452-454 (1985)] by splicing the genes from a mouse antibody moleculespecific for an ob polypeptide together with genes from a human antibodymolecule of appropriate biological activity can be used; such antibodiesare within the scope of this invention. Such human or humanized chimericantibodies are preferred for use in therapy of human diseases ordisorders (described infra), since the human or humanized antibodies aremuch less likely than xenogenic antibodies to induce an immune response,in particular an allergic response, themselves.

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778) can be adapted toproduce OB polypeptide-specific single-chain antibodies. An additionalembodiment of the invention utilizes the techniques described for theconstruction of Fab expression libraries [Huse et al., Science,246:1275-1281 (1989)] to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity for an OBpolypeptide, or its derivatives, or analogs.

Antibody fragments which contain the idiotype of the antibody moleculecan be generated by known techniques. For example, such fragmentsinclude but are not limited to: the F(ab')₂ fragment which can beproduced by pepsin digestion of the antibody molecule; the Fab'fragmentswhich can be generated by reducing the disulfide bridges of the F(ab')₂fragment, and the Fab fragments which can be generated by treating theantibody molecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g., radioimnmunoassay,ELISA (enzyme-linked immunosorbent assay), "sandwich" imnmunoassays,immunoradiometric assays, gel diffusion precipitin reactions,immnunodiffusion assays, in situ immunoassays (using colloidal gold,enzyme or radioisotope labels, for example), Western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention. For example, to select antibodies which recognize aspecific epitope of an OB polypeptide, one may assay generatedhybridomas for a product which binds to an OB polypeptide fragmentcontaining such epitope. For selection of an antibody specific to an OBpolypeptide from a particular species of animal, one can select on thebasis of positive binding with OB polypeptide expressed by or isolatedfrom cells of that species of animal.

The foregoing antibodies can be used in methods known in the artrelating to the localization and activity of the OB polypeptide, e.g.,for Western blotting, imaging OB polypeptide in situ, measuring levelsthereof in appropriate physiological samples, etc. In a specificembodiment, antibodies that agonize or antagonize the activity of OBpolypeptide can be generated. Such antibodies can be tested using theassays described infra for identifying ligands.

In a specific embodiment, antibodies are developed by immunizing rabbitswith synthetic peptides predicted by the protein sequence or withrecombinant proteins made using bacterial expression vectors. The choiceof synthetic peptides is made after careful analysis of the predictedprotein structure, as described above. In particular, peptide sequencesbetween putative cleavage sites are chosen. Synthetic peptides areconjugated to a carrier such as KLH hemocyanin or BSA using carbodiimideand used in Freund's adjuvant to immunize rabbits. In order to preparerecombinant protein, the pGEX vector can be used to express thepolypeptide (Smith et al., 1988, supra). Alternatively, one can use onlyhydrophilic domains to generate the fusion protein. The expressedprotein will be prepared in quantity and used to immunize rabbits inFreund's adjuvant.

In another specific embodiment, recombinant OB polypeptide is used toimmunize chickens, and the chicken anti-OB antibodies are recovered fromegg yolk, e.g., by affinity purification on an OB-column. Preferably,chickens used in immunization are kept under specific pathogen free(SPF) conditions.

In another embodiment, antibodies against leptin are generated in ob/obmice, which lack circulating OB protein, and thus are expected to becapable of generating an anti-OB polypeptide response since they willnot be tolerized to the polypeptide, and wild-type mice. Spleen cellsfrom both groups of mice can be fused with myeloma cells to preparehybridomas for monoclonal antibodies.

In yet another embodiment, recombinant OB polypeptide is used toimmunize rabbits, and the polyclonal antibodies are immunopurified priorto further use. The purified antibodies are particularly useful forsemi-quantitative assays, particularly for detecting the presence ofcirculating OB polypeptide in serum or plasma.

Panels of monoclonal antibodies produced against modulator peptides canbe screened for various properties; i.e., isotype, epitope, affinity,etc. Of particular interest are monoclonal antibodies that neutralizethe activity of the modulator peptides. Such monoclonals can be readilyidentified in activity assays for the weight modulators. High affinityantibodies are also useful when immunoaffmity purification of native orrecombinant modulator is possible.

Preferably, the anti-modulator antibody used in the diagnostic andtherapeutic methods of this invention is an affinity-purified polyclonalantibody. More preferably, the antibody is a monoclonal antibody (niAb).In addition, it is preferable for the anti-modulator antibody moleculesused herein be in the form of Fab, Fab', F(ab')₂ or F(v) portions ofwhole antibody molecules.

Diagnostic Implications

The present invention also relates to a variety of diagnosticapplications, including methods for detecting the presence of conditionsand/or stimuli that impact upon abnormalities in body weight oradiposity, by reference to their ability to elicit the activities whichare mediated by the present weight modulators. As mentioned earlier, theweight modulator peptides can be used to produce antibodies tothemselves by a variety of known techniques, and such antibodies couldthen be isolated and utilized as in tests for the presence of particulartranscriptional activity in suspect target cells. Alternatively, thenucleic acids of the invention can be employed in diagnosis.

Antibody-based Diagnostics

As suggested earlier, a diagnostic method useful in the presentinvention comprises examining a cellular sample or medium by means of anassay including an effective amount of an antagonist to a modulatorprotein, such as an anti-modulator antibody, preferably anaffinity-purified polyclonal antibody, and more preferably a mAb. Inaddition, it is preferable for the anti-modulator antibody moleculesused herein be in the form of Fab, Fab', F(ab')₂ or F(v) portions orwhole antibody molecules. As previously discussed, patients capable ofbenefiting from this method include those suffering from cancer, AIDS,obesity or other conditions where abnormal body weight is acharacteristic or factor. Methods for isolating the modulator andinducing anti-modulator antibodies and for determining and optimizingthe ability of anti-modulator antibodies to assist in the examination ofthe target cells are all well-known in the art.

Also, antibodies including both polyclonal and monoclonal antibodies,and drugs that modulate the production or activity of the weight controlmodulators and other recognition factors and/or their subunits maypossess certain diagnostic applications and may for example, be utilizedfor the purpose of detecting and/or measuring conditions whereabnormalities in body weight are or may be likely to develop. Forexample, the modulator peptides or their active fragments may be used toproduce both polyclonal and monoclonal antibodies to themselves in avariety of cellular media, by known techniques, such as the hybridomatechnique utilizing, for example, fused mouse spleen lymphocytes andmyeloma cells. These techniques are described in detail below. Likewise,small molecules that mimic or antagonize the activity(ies) of thereceptor recognition factors of the invention may be discovered orsynthesized, and may be used in diagnostic and/or therapeutic protocols.

The presence of weight modulators in cells can be ascertained by theusual immunological procedures applicable to such determinations. Anumber of useful procedures are known. Three such procedures which areespecially useful utilize either the receptor recognition factor labeledwith a detectable label, antibody Ab₁ labeled with a detectable label,or antibody Ab₂ labeled with a detectable label. The procedures may besummarized by the following equations wherein the asterisk indicatesthat the particle is labeled, and "WM" stands for the weight modulator:

    A. WM*+Ab.sub.1 =WM*Ab.sub.1

    B. WM+Ab.sub.* =WMAb.sub.1 *

    C. WM+Ab.sub.1 +Ab.sub.2 *=Ab.sub.1 WMAb.sub.2 *

The procedures and their application are all familiar to those skilledin the art and accordingly may be utilized within the scope of thepresent invention. The "competitive" procedure, Procedure A, isdescribed in U.S. Pat. Nos. 3,654,090 and 3,850,752. Procedure B isrepresentative of well known competitive assay techniques. Procedure C,the "sandwich" procedure, is described in U.S. Pat. Nos. RE31,006 and4,016,043. Still other procedures are known such as the "doubleantibody", or "DASP" procedure.

In each instance, the weight modulators form complexes with one or moreantibody(ies) or binding partners and one member of the complex islabeled with a detectable label. The fact that a complex has formed and,if desired, the amount thereof, can be determined by known methodsapplicable to the detection of labels.

It will be seen from the above, that a characteristic property of Ab₂ isthat it will react with Ab₁. This is because Ab₁, raised in onemammalian species, has been used in another species as an antigen toraise the antibody, Ab₂. For example, Ab₂ may be raised in goats usingrabbit antibodies as antigens. Ab₂ therefore would be anti-rabbitantibody raised in goats. For purposes of this description and claims,Ab₁ will be referred to as a primary or anti-weight modulator antibody,and Ab₂ will be referred to as a secondary or anti-Ab₁ antibody.

The labels most commonly employed for these studies are radioactiveelements, enzymes, chemicals which fluoresce when exposed to ultravioletlight, and others.

A number of fluorescent materials are known and can be utilized aslabels. These include, for example, fluorescein, rhodamine and auramine.A particular detecting material is anti-rabbit antibody prepared ingoats and conjugated with fluorescein through an isothiocyanate.

The weight modulators or their binding partners can also be labeled witha radioactive element or with an enzyme. The radioactive label can bedetected by any of the currently available counting procedures. Thepreferred isotope may be selected from ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, and ¹⁸⁶ Re.

Enzyme labels are likewise useful, and can be detected by any of thepresently utilized colorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques. Theenzyme is conjugated to the selected particle by reaction with bridgingmolecules such as carbodiimides, diisocyanates, glutaraldehyde and thelike. Many enzymes which can be used in these procedures are known andcan be utilized. The preferred are peroxidase, β-glucuronidase,β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plusperoxidase and alkaline phosphatase. U.S. Pat. Nos. 3,654,090;3,850,752; and 4,016,043 are referred to by way of example for theirdisclosure of alternate labeling material and methods.

In a further embodiment of this invention, test kits suitable for use bya medical specialist may be prepared to determine the presence orabsence of predetermined transcriptional activity or predeterminedtranscriptional activity capability of OB in suspected target cells. Inaccordance with the testing techniques discussed above, one class ofsuch kits will contain at least the labeled weight modulator or itsbinding partner, for instance an antibody specific thereto, anddirections, of course, depending upon the method selected, e.g.,"competitive," "sandwich," "DASP" and the like. The kits may alsocontain peripheral reagents such as buffers, stabilizers, etc.

Accordingly, a test kit may be prepared for the demonstration of thepresence or capability of cells for predetermined transcriptionalactivity, comprising:

(a) a predetermined amount of at least one labeled immunochemicallyreactive component obtained by the direct or indirect attachment of thepresent weight modulator or a specific binding partner thereto, to adetectable label;

(b) other reagents; and

(c) directions for use of said kit.

More specifically, the diagnostic test kit may comprise:

(a) a known amount of the weight modulator as described above (or abinding partner) generally bound to a solid phase to form animmunosorbent, or in the alternative, bound to a suitable tag, or pluralsuch end products, etc. (or their binding partners) one of each;

(b) if necessary, other reagents; and

(c) directions for use of said test kit.

In a further variation, the test kit may be prepared and used for thepurposes stated above, which operates according to a predeterminedprotocol (e.g. "competitive," "sandwich," "double antibody," etc.), andcomprises:

(a) a labeled component which has been obtained by coupling the weightmodulator to a detectable label;

(b) one or more additional immunochemical reagents of which at least onereagent is a ligand or an immobilized ligand, which ligand is selectedfrom the group consisting of:

(i) a ligand capable of binding with the labeled component (a);

(ii) a ligand capable of binding with a binding partner of the labeledcomponent (a);

(iii) a ligand capable of binding with at least one of the component(s)to be determined; and

(iv) a ligand capable of binding with at least one of the bindingpartners of at least one of the component(s) to be determined; and

(c) directions for the performance of a protocol for the detectionand/or determination of one or more components of an immunochemicalreaction between the weight modulator and a specific binding partnerthereto.

Nucleic Acid-based Diagnostics

As demonstrated in the examples, infra, nucleic acids of the inventioncan be used to detect defects associated with defects in the OBpolypeptide that result in obese phenotypes. For example, nucleic acidprobes (e.g., in Northern analysis or RT-PCR analysis) can be used todetermine whether an obese phenotype is associated with lack ofexpression of OB mRNA, or expression of non-functional OB mRNA, e.g., asin db/db mice (where the deficiency results from lack of an OB receptor)or where a mutation yields a non-transcribed mRNA. Moreover, the nucleicacid-based diagnostic techniques of the invention can be used inconjunction with antibody-based techniques to further develop amolecular understanding of obese or anorexic phenotypes.

The human cDNA clones that have recently been isolated have beensequenced as presented herein. This facilitates the determination of thecomplete sequence of the human gene (see FIG. 20A through C; SEQ IDNO:22). DNA sequences from the introns of the human OB gene have beenobtained (FIG. 20), and these have been used to prepare PCR primers toPCR amplify the coding sequence of the OB gene from human genomic DNA soas to identify mutations or allelic variants of the OB gene, all inaccordance with protocols described in detail earlier herein. SpecificPCR primers for amplifying human genomic OB are described in a specificExample, infra.

The current hypothesis is that heterozygous mutations in the ob genewill be associated with mild/moderate obesity while homozygous mutationswould be associated with several DNA sequence-based diagnostic tests forobesity. If this is true, it would allow the ascertainment of people atrisk for the development of obesity and make possible the application ofdrug treatment and/or lifestyle changes before an increased body weightis fully developed.

Alternatively, the presence of microsatellites that segregate withmutant forms of human OB can be used for diagnosis. Various PCR primers,including those based on the nucleotide sequence provided in FIG. 20Athrough C, can be used in this respect.

The OB gene may also be useful diagnostically for measurements of itsencoded RNA and protein in nutritional disorders. It will be ofimportance to know, in a particular nutritional disorder, whether OB RNAand/or its encoded protein is unregulated or downregulated. Thus, if anobese person has increased levels of OB, it would appear that theproblem is downstream of OB, while if OB is reduced, it would appearthat inappropriately low levels of OB may be cause of obesity (whetheror not the defect is in the OB gene). Conversely, if a cancer or AIDSpatient who lost weight had elevated levels of OB, it may be concludedthat inappropriately high expression of OB is responsible for the weightloss.

The cloned human cDNA will be of use for the measurement of the levelsof human OB RNA. In addition, recombinant human protein will be preparedand used to develop immunoassays to enable measurement of the fat andperhaps plasma levels of the OB protein.

Therapeutic Implications

The polypeptides, nucleic acids, and antibodies of the invention havesignificant therapeutic potential. Preferably, a therapeuticallyeffective amount of such an agent is administered in a pharmaceuticallyacceptable carrier, diluent, or excipient.

The phrase "pharmaceutically acceptable" refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similarly untoward reaction, such as gastricupset, dizziness and the like, when administered to a human. Preferably,as used herein, the term "pharmaceutically acceptable" means approved bya regulatory agency of the federal or a state government or listed inthe U.S. Pharmacopeia or other generally recognized phamacopeia for usein animals, and more particularly in humans. The term "carrier" refersto a diluent, adjuvant, excipient, or vehicle with which the compound isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water or solution saline solutions and aqueousdextrose and glycerol solutions are preferably employed as carriers,particularly for injectable solutions. Suitable pharmaceutical carriersare described in Martin, Remington's Pharmaceutical Sciences, 18th Ed.,Mack Publishing Co., Easton, Pa., (1990).

The phrase "therapeutically effective amount" is used herein to mean anamount sufficient to reduce by at least about 15%, preferably by atleast 50%, more preferably by at least 90%, and most preferably prevent,a clinically significant deficit in the activity, function and responseof the host. Alternatively, a therapeutically effective amount issufficient to cause an improvement in a clinically significant conditionin the host.

Administration of recombinant OB polypeptide results in weight loss, inparticular, a decrease in fat tissue. OB polypeptide can be preparedusing standard bacterial and/or mammalian expression vectors,synthetically, or purified from plasma or serum, all as stated in detailearlier herein. Alternatively, increased expression of native OBpolypeptide may be induce by homologous recombination techniques, asdescribed supra.

Reduction of OB polypeptide activity (by developing antagonists,inhibitors, use of neutralizing antibodies, or antisense molecules)should result in weight gain as might be desirable for the treatment ofthe weight loss associated with cancer, AIDS or anorexia nervosa.Modulation of OB activity can be useful for reducing body weight (byincreasing its activity) or increasing body weight (by decreasing itsactivity).

Polypeptide-based Therapeutic Treatment

In the simplest analysis, the OB gene determines body weight in mammals,in particular, mice and man. The OB gene product, and, correspondingly,cognate molecules, appear to be part of a signaling pathway by whichadipose tissue communicates with the brain and the other organs. It isbelieved that the OB polypeptide is itself a signaling molecule, i.e., ahormone.

The OB polypeptide, or functionally active fragment thereof, or anantagonist thereof, can be administered orally or parenterally,preferably parenterally. Because metabolic homeostasis is a continuousprocess, controlled release administration of OB polypeptide ispreferred. For example, the polypeptide may be administered usingintravenous infusion, an implantable osmotic pump, a transdermal patch,liposomes, or other modes of administration. In one embodiment, a pumpmay be used [Langer et al., eds., Medical Applications of ControlledRelease, CRC Pres., Boca Raton, Fla. (1974); Sefton, CRC Crit. RefBiomed. Eng., 14:201 (1987); Buchwald et al., Surgery, 88:507 (1980);Saudek et al., N. Engl. J. Med., 321:574 (1989)]. In another embodiment,polymeric materials can be used [see, Langer, 1974, supra; Sefton, 1987,supra; Smolen et al., eds., Controlled Drug Bioavailability, DrugProduct Design and Performance, Wiley, N.Y. (1984); Ranger et al., J.Macromol. Sci. Rev. Macromol. Chem., 23:61 (1983); see also Levy et al.,Science, 228:190 (1985); During et al., Ann. Neurol., 25:351 (1989);Howard et al., J. Neurosurg., 71:105 (1989)]. In yet another embodiment,a controlled release system can be placed in proximity of thetherapeutic target, i.e., the brain, thus requiring only a fraction ofthe systemic dose [see, e.g., Goodson, in Medical Applications ofControlled Release, vol. 2, pp. 115-138 (1984)]. Other controlledrelease systems are discussed in the review by Langer, Science,249:1527-1533 (1990). In another embodiment, the therapeutic compoundcan be delivered in a vesicle, in particular a liposome (see Langer,1990, supra); Treat et al., in Liposomes in the Therapy of InfectiousDisease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, N.Y., pp.353-365 (1989); Lopez-Berestein, pp. 317-327; see generally ibid.)

In a further aspect, recombinant cells that have been transformed withthe OB gene and that express high levels of the polypeptide can betransplanted in a subject in need of OB polypeptide. Preferablyautologous cells transformed with OB are transplanted to avoidrejection; alternatively, technology is available to shieldnon-autologous cells that produce soluble factors within a polymermatrix that prevents immune recognition and rejection.

The OB polypeptide can be delivered by intravenous, intraarterial,intraperitoneal, intramuscular, or subcutaneous routes ofadministration. Alternatively, the OB polypeptide, properly formulated,can be administered by nasal or oral administration. A constant supplyof OB can be ensured by providing a therapeutically effective dose(i.e., a dose effective to induce metabolic changes in a subject) at thenecessary intervals, e.g., daily, every 12 hours, etc. These parameterswill depend on the severity of the disease condition being treated,other actions, such as diet modification, that are implemented, theweight, age, and sex of the subject, and other criteria, which can bereadily determined according to standard good medical practice by thoseof skill in the art.

Pharmaceutical Compositions

In yet another aspect of the present invention, provided arepharmaceutical compositions of the above. Such pharmaceuticalcompositions may be for administration for injection, or for oral,pulmonary, nasal or other forms of administration. In general,comprehended by the invention are pharmaceutical compositions comprisingeffective amounts of protein or derivative products of the inventiontogether with pharmaceutically acceptable diluents, preservatives,solubilizers, emulsifiers, adjuvants and/or carriers. Such compositionsinclude diluents of various buffer content (e.g., Tris-HCl, acetate,phosphate), pH and ionic strength; additives such as detergents andsolubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants(e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g.,Thimersol, benzyl alcohol) and bulking substances (e.g., lactose,mannitol); incorporation of the material into particulate preparationsof polymeric compounds such as polylactic acid, polyglycolic acid, etc.or into liposomes. Hylauronic acid may also be used. Such compositionsmay influence the physical state, stability, rate of in vivo release,and rate of in vivo clearance of the present proteins and derivatives.See, e.g., Martin, pp.1435-1712, 1990, supra, which are hereinincorporated by reference. The compositions may be prepared in liquidform, or may be in dried powder, such as lyophilized form.

Oral Delivery

Contemplated for use herein are oral solid dosage forms, which aredescribed generally in Martin, Chapter 89, 1990, supra, which is hereinincorporated by reference. Solid dosage forms include tablets, capsules,pills, troches or lozenges, cachets or pellets. Also, liposomal orproteinoid encapsulation may be used to formulate the presentcompositions (as, for example, proteinoid microspheres reported in U.S.Pat. No. 4,925,673). Liposomal encapsulation may be used and theliposomes may be derivatized with various polymers (e.g., U.S. Pat. No.5,013,556). A description of possible solid dosage forms for thetherapeutic is given by Marshall, in Modern Pharmaceutics, Chapter 10,Banker and Rhodes ed., (1979), herein incorporated by reference. Ingeneral, the formulation will include the protein (or chemicallymodified protein), and inert ingredients which allow for protectionagainst the stomach environment, and release of the biologically activematerial in the intestine.

Also specifically contemplated are oral dosage forms of the abovederivatized proteins. Protein may be chemically modified so that oraldelivery of the derivative is efficacious. Generally, the chemicalmodification contemplated is the attachment of at least one moiety tothe protein (or peptide) molecule itself, where said moiety permits (a)inhibition of proteolysis; and (b) uptake into the blood stream from thestomach or intestine. Also desired is the increase in overall stabilityof the protein and increase in circulation time in the body. Examples ofsuch moieties include: polyethylene glycol, copolymers of ethyleneglycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinylalcohol, polyvinyl pyrrolidone and polyproline. Abuchowski et al., 1981,supra; Newmark et al., J. Appl. Biochem., 4:185-189 (1982). Otherpolymers that could be used are poly-1,3-dioxolane andpoly-1,3,6-tioxocane. Preferred for pharmaceutical usage, as indicatedabove, are polyethylene glycol moieties.

For the protein (or derivative) the location of release may be thestomach, the small intestine (the duodenum, the jejunem, or the ileum),or the large intestine. One skilled in the art has availableformulations which will not dissolve in the stomach, yet will releasethe material in the duodenum or elsewhere in the intestine. Preferably,the release will avoid the deleterious effects of the stomachenvironment, either by protection of the protein (or derivative) or byrelease of the biologically active material beyond the stomachenvironment, such as in the intestine.

To ensure full gastric resistance, a coating impermeable to at least pH5.0 is essential. Examples of the more common inert ingredients that areused as enteric coatings are cellulose acetate trimellitate (CAT),hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55,polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, celluloseacetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. Thesecoatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which arenot intended for protection against the stomach. This can include sugarcoatings, or coatings which make the tablet easier to swallow. Capsulesmay consist of a hard shell (such as gelatin) for delivery of drytherapeutic i.e. powder; for liquid forms, a soft gelatin shell may beused. The shell material of cachets could be thick starch or otheredible paper. For pills, lozenges, molded tablets or tablet triturates,moist massing techniques can be used.

The therapeutic can be included in the formulation as finemultiparticulates in the form of granules or pellets of particle sizeabout 1 mm. The formulation of the material for capsule administrationcould also be as a powder, lightly compressed plugs or even as tablets.The therapeutic could be prepared by compression.

Colorants and flavoring agents may all be included. For example, theprotein (or derivative) may be formulated (such as by liposome ormicrosphere encapsulation) and then further contained within an edibleproduct, such as a refrigerated beverage containing colorants andflavoring agents.

One may dilute or increase the volume of the therapeutic with an inertmaterial. These diluents could include carbohydrates, especiallymannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modifieddextrans and starch. Certain inorganic salts may be also be used asfillers including calcium triphosphate, magnesium carbonate and sodiumchloride. Some commercially available diluents are Fast-Flo, Emdex,STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic intoa solid dosage form. Materials used as disintegrants include but are notlimited to starch including the commercial disintegrant based on starch,Explotab. Sodium starch glycolate, Amberlite, sodiumcarboxymethylcellulose, ultramylopectin, sodium alginate, gelatin,orange peel, acid carboxymethyl cellulose, natural sponge and bentonitemay all be used. Another form of the disintegrants are the insolublecationic exchange resins. Powdered gums may be used as disintegrants andas binders and these can include powdered gums such as agar, Karaya ortragacanth. Alginic acid and its sodium salt are also useful asdisintegrants.

Binders may be used to hold the therapeutic agent together to form ahard tablet and include materials from natural products such as acacia,tragacanth, starch and gelatin. Others include methyl cellulose (MC),ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both beused in alcoholic solutions to granulate the therapeutic.

An antifrictional agent may be included in the formulation of thetherapeutic to prevent sticking during the formulation process.Lubricants may be used as a layer between the therapeutic and the diewall, and these can include but are not limited to: stearic acidincluding its magnesium and calcium salts, polytetrafluoroethylene(PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricantsmay also be used such as sodium lauryl sulfate, magnesium laurylsulfate, polyethylene glycol of various molecular weights, and Carbowax4000 and 6000.

Glidants that might improve the flow properties of the drug duringformulation and to aid rearrangement during compression might be added.The glidants may include starch, talc, pyrogenic silica and hydratedsilicoaluminate.

To aid dissolution of the therapeutic into the aqueous environment, asurfactant might be added as a wetting agent. Surfactants may includeanionic detergents such as sodium lauryl sulfate, dioctyl sodiumsulfosuccinate and dioctyl sodium sulfonate. Cationic detergents mightbe used and could include benzalkonium chloride or benzethomiumchloride. The list of potential nonionic detergents that could beincluded in the formulation as surfactants are lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fattyacid ester, methyl cellulose and carboxymethyl cellulose. Thesesurfactants could be present in the formulation of the protein orderivative either alone or as a mixture in different ratios.

Additives which potentially enhance uptake of the protein (orderivative) are for instance the fatty acids oleic acid, linoleic acidand linolenic acid.

Controlled release formulation may be desirable. The drug could beincorporated into an inert matrix which permits release by eitherdiffusion or leaching mechanisms i.e., gums. Slowly degeneratingmatrices may also be incorporated into the formulation. Another form ofa controlled release of this therapeutic is by a method based on theOros therapeutic system (Alza Corp.), i.e. the drug is enclosed in asemipermeable membrane which allows water to enter and push the drug outthrough a single small opening due to osmotic effects. Some entericcoatings also have a delayed release effect.

Other coatings may be used for the formulation. These include a varietyof sugars which could be applied in a coating pan. The therapeutic agentcould also be given in a film-coated tablet; the materials used in thisinstance are divided into 2 groups. The first are the nonentericmaterials and include methyl cellulose, ethyl cellulose, hydroxyethylcellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose,hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose,providone and the polyethylene glycols. The second group consists of theenteric materials that are commonly esters of phthalic acid.

A mix of materials might be used to provide the optimum film coating.Film coating may be carried out in a pan coater or in a fluidized bed orby compression coating.

Pulmonary Delivery

Also contemplated herein is pulmonary delivery of the present protein(or derivatives thereof). The protein (or derivative) is delivered tothe lungs of a mammal while inhaling and traverses across the lungepithelial lining to the blood-stream. Other reports of this includeAdjei et al., Pharmaceutical Research, 7(6):565-569 (1990); Adjei etal., International Journal of Pharmaceutics, 63:135-144 (1990)(leuprolide acetate); Braquet et al., Journal of CardiovascularPharmacology, 13(suppl. 5):143-146 (1989) (endothelin-1); Hubbard etal., Annals of Internal Medicine, 3(3):206-212 (1989) (α1- antitrypsin);Smith et al., J. Clin. Invest., 84:1145-1146 (1989) (α1-proteinase);Oswein et al., "Aerosolization of Proteins", Proceedings of Symposium onRespiratory Drug Delivery II, Keystone, Colo., (March 1990) (recombinanthuman growth hormone); Debs et al., J. Immunol., 140:3482-3488 (1988)(interferon-γ and tumor necrosis factor alpha) and Platz et al., U.S.Pat. No. 5,284,656 (granulocyte colony stimulating factor). Contemplatedfor use in the practice of this invention are a wide range of mechanicaldevices designed for pulmonary delivery of therapeutic products,including but not limited to nebulizers, metered-dose inhalers, andpowder inhalers, all of which are familiar to those skilled in the art.

Some specific examples of commercially available devices suitable forthe practice of this invention are the Ultravent nebulizer, manufacturedby Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer,manufactured by Marquest Medical Products, Englewood, Colo.; theVentolin metered-dose inhaler, manufactured by Glaxo Inc., ResearchTriangle Park, N.C.; and the Spinhaler powder inhaler, manufactured byFisons Corp., Bedford, Mass.

All such devices require the use of formulations suitable for thedispensing of protein (or derivative). Typically, each formulation isspecific to the type of device employed and may involve the use of anappropriate propellant material, in addition to the usual diluents,adjuvants and/or carriers useful in therapy. Also, the use of liposomes,microcapsules or microspheres, inclusion complexes, or other types ofcarriers is contemplated. Chemically modified protein may also beprepared in different formulations depending on the type of chemicalmodification or the type of device employed.

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise protein (or derivative) dissolved inwater at a concentration of about 0.1 to 25 mg of biologically activeprotein per ml of solution. The formulation may also include a bufferand a simple sugar (e.g., for protein stabilization and regulation ofosmotic pressure). The nebulizer formulation may also contain asurfactant, to reduce or prevent surface induced aggregation of theprotein caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the protein (or derivative)suspended in a propellant with the aid of a surfactant. The propellantmay be any conventional material employed for this purpose, such as achlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise afmely divided dry powder containing protein (or derivative) and may alsoinclude a bulking agent, such as lactose, sorbitol, sucrose, or mannitolin amounts which facilitate dispersal of the powder from the device,e.g., 50 to 90% by weight of the formulation. The protein (orderivative) should most advantageously be prepared in particulate formwith an average particle size of less than 10 μm (or microns), mostpreferably 0.5 to 5 μm, for most effective delivery to the distal lung.

Nasal Delivery

Nasal delivery of the protein (or derivative) is also contemplated.Nasal delivery allows the passage of the protein to the blood streamdirectly after administering the therapeutic product to the nose,without the necessity for deposition of the product in the lung.Formulations for nasal delivery include those with dextran orcyclodextran.

Methods of Treatment, Methods of Preparing a Medicament

In yet another aspect of the present invention, methods of treatment andmanufacture of a medicament are provided. Conditions alleviated by ormodulated by the administration of the present derivatives are thoseindicated above.

Dosages

For all of the above molecules, as further studies are conducted,information will emerge regarding appropriate dosage levels fortreatment of various conditions in various patients, and the ordinaryskilled worker, considering the therapeutic context, age and generalhealth of the recipient, will be able to ascertain the proper dosage.Generally, for injection or infusion, dosage will be between 0.01 μg ofbiologically active protein/kg body weight, (calculating the mass of theprotein alone, without chemical modification), and 10 mg/kg (based onthe same). The dosing schedule may vary, depending on the circulationhalf-life of the protein or derivative used, whether the polypeptide isdelivered by bolus dose or continuous infusion, and the formulationused.

Administration with other compounds

For therapy associated with obesity, one may administer the presentprotein (or derivatives) in conjunction with one or more pharmaceuticalcompositions used for treating other clinical complications of obesity,such as those used for treatment of diabetes (e.g., insulin), high bloodpressure, high cholesterol, and other adverse conditions incident toobesity. Also, other appetite suppressants may be co-administered, e.g.,amphetamines. Administration may be simultaneous (for example,administration of a mixture of the present protein and insulin) or maybe in seriatim.

Nucleic Acid-based Therapeutic Treatment

The OB gene could be introduced into human fat cells to develop genetherapy for obesity. Such therapy would be expected to decrease bodyweight. Conversely, introduction of antisense constructs into human fatcells would reduce the levels of active OB polypeptide and would bepredicted to increase body adiposity.

In one embodiment, a gene encoding an OB polypeptide is introduced invivo in a viral vector. Such vectors include an attenuated or defectiveDNA virus, such as but not limited to herpes simplex virus (HSV),papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associatedvirus (AAV), and the like. Defective viruses, which entirely or almostentirely lack viral genes, are preferred. Defective virus is notinfective after introduction into a cell. Use of defective viral vectorsallows for administration to cells in a specific, localized area,without concern that the vector can infect other cells. Thus, adiposetissue can be specifically targeted. Examples of particular vectorsinclude, but are not limited to, a defective herpes virus 1 (HSV1)vector [Kaplitt et al., Molec. Cell. Neurosci., 2:320-330 (1991)], anattenuated adenovirus vector, such as the vector described byStratford-Perricaudet et al., J. Clin. Invest., 90:626-630 (1992), and adefective adeno-associted virus vector [Samulski et al., J. Virol.,61:3096-3101 (1987); Samulski et al., J. Virol., 63:3822-3828 (1989)].

In another embodiment, the gene can be introduced in a retroviralvector, e.g., as described in Anderson et al., U.S. Pat. No. 5,399,346;Mann et al., Cell, 33:153 (1983); Temin et al., U.S. Pat. No. 4,650,764;Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J. Virol.,62:1120 (1988); Temin et al., U.S. Pat. No. 5,124,263; InternationalPatent Publication No. WO 95/07358, published Mar. 16, 1995, byDougherty et al.; and Kuo et al., Blood, 82:845 (1993).

Alternatively, the vector can be introduced in vivo by lipofection. Forthe past decade, there has been increasing use of liposomes forencapsulation and transfection of nucleic acids in vitro. Syntheticcationic lipids designed to limit the difficulties and dangersencountered with liposome mediated transfection can be used to prepareliposomes for in vivo transfection of a gene encoding a marker [Felgneret al., Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987); see Mackey etal., Proc. Natl. Acad. Sci. USA, 85:8027-8031 (1988)]. The use ofcationic lipids may promote encapsulation of negatively charged nucleicacids, and also promote fusion with negatively charged cell membranes[Felgner et al., Science, 337:387-388 (1989)]. The use of lipofection tointroduce exogenous genes into specific organs in vivo has certainpractical advantages. Molecular targeting of liposomes to specific cellsrepresents one area of benefit. It is clear that directing transfectionto particular cell types would be particularly advantageous in a tissuewith cellular heterogeneity, such as the pancreas, liver, kidney, andbrain. Lipids may be chemically coupled to other molecules for thepurpose of targeting (see Mackey et al., 1988, supra). Targetedpeptides, e.g., hormones or neurotransmitters, and proteins such asantibodies, or non-peptide molecules could be coupled to liposomeschemically.

It is also possible to introduce the vector in vivo as a naked DNAplasmid. Naked DNA vectors for gene therapy can be introduced into thedesired host cells by methods known in the art, e.g., transfection,electroporation, microinjection, transduction, cell fusion, DEAEdextran, calcium phosphate precipitation, use of a gene gun, or use of aDNA vector transporter (see, e.g., Wu et al., J. Biol. Chem.,267:963-967 (1992); Wu et al., J. Biol. Chem., 263:14621-14624 (1988);Hartmut et al., Canadian Patent Application No. 2,012,311, filed Mar.15, 1990).

Agricultural Applications

The OB gene can also be isolated from domestic animals, and thecorresponding OB polypeptide obtained thereby. In a specific example,infra, the probe derived from the murine OB gene hybridizes tocorresponding homologous coding sequences from a large number of speciesof animals. As discussed for human therapies, recombinant proteins canalso be prepared and administered to domestic animals. Administration ofthe polypeptide can be implemented to produce leaner food animals, suchas beef cattle, swine, poultry, sheep, etc. Preferably, an autologous OBpolypeptide is administered, although the invention contemplatesadministration of anti-autologous polypeptide as well. Since the OBpolypeptide consists of approximately 160 amino acid residues, it maynot be highly immunogenic. Thus, administration of non-autologouspolypeptide may not result in an immune response.

Alternatively, the introduction of the cloned genes into transgenicdomestic animals would allow one to potentially decrease body weight andadiposity by overexpressing an OB transgene. The simplest means ofachieving this would be to target an OB transgene to fat using its ownor another fat specific promoter.

Conversely, increases in body fat might be desirable in othercircumstances such as for the development of Kobe beef or fatty liver tomake foie gras. This could be accomplished by targeting an antisense OBtransgene to fat, or by using gene knockout technology. Alternatively,where an increase in body weight at percentage of fat is desired, aninhibitor or antagonist of the OB polypeptide can be administered. Suchinhibitors or antagonists include, but are not limited to, antibodiesreactive with the polypeptide, and fragments of the polypeptide thatbind but do not activate the OB receptor, i.e., antagonists of the OBpolypeptide.

Cosmetic Implications

The OB polypeptide has significant value for cosmetic use, in additionto the health benefits. In particular, since the OB polypeptides of theinvention, including derivatives and agonist analogs thereof, are usefulfor modulation of the rate and quantity of fat cell deposition in ananimal, they are useful for reducing unsightly fat tissue, e.g., fatdeposits in the abdomen, hips, thighs, neck, and chin that do notnecessarily amount to an obese condition, but which nevertheless detractfrom an individual's appearance. The fat reduction effect is thought tobe accomplished, in part, by a reduction in appetite, i.e., a reductionin food intake, by an increase in basal metabolism, or both. Thus, thepresent OB polypeptide, or its derivatives or agonist analogs, is usefulfor administration to a subject to effect cosmetic changes in fat tissuedeposits, whether by modulating fat deposition, reducing appetite, orboth.

In addition, the present compositions and methods may be used inconjunction with various procedures, such as cosmetic surgeries designedto alter the overall appearance of a body (e.g., liposuction or lasersurgeries designed to reduce body mass by aspirating or ablating fattissue), exercise (especially running and weight training), low fatdiet, hypnosis, biofeedback, as examples of the ways one may attempt todecrease the percentage of fat tissue and improve the appearance of thebody.

Accordingly, the present invention relates to a method for effectingcosmetic fat tissue modulation in an individual comprising administeringa fat modulating amount of an OB polypeptide, or derivative or agonistanalog thereof, to an individual who desires cosmetic fat tissuemodulation to improve overall body appearance. In a particular aspect,the fat tissue modulation is a consequence of appetite suppression.Preferably, the fat tissue modulation is a reduction in fat tissue.

In a further embodiment, the invention relates to a method for effectingcosmetic fat tissue loss comprising combining a procedure for changingbody appearance with administration of a fat modulating amount of an OBpolypeptide, or derivative or agonist analog thereof, to an individualwho desires cosmetic fat tissue modulation to improve overall bodyappearance.

The OB Receptor

Development of small molecule agonists and antagonists of the OB factorwill be greatly facilitated by the isolation of its receptor. This canbe accomplished by preparing active OB polypeptide and using it toscreen an expression library using standard methodology. Receptorbinding in the expression library can be tested by administeringrecombinant polypeptide prepared using either bacterial or mammalianexpression vectors, and observing the effects of short term andcontinuous administration of the recombinant polypeptide on the cells ofthe expression library, or by directly detecting binding of OBpolypeptide to the cells.

As it is presently believed that the OB receptor is likely to be locatedin the hypothalamus and perhaps liver, preferably cDNA libraries fromthese tissues will be constructed in standard expression cloningvectors. These cDNA clones would next be introduced into COS cells aspools and the resulting transformants would be screened with activeligand to identify COS cells expressing the OB receptor. Positive clonescan then be isolated so as to recover the cloned receptor. The clonedreceptor would be used in conjunction with the OB ligand (assuming it isa hormone) to develop the necessary components for screening of smallmolecule modulators of OB.

A particular assay system that is to be utilized in accordance with thepresent invention, is known as a receptor assay. In a receptor assay,the material to be assayed is appropriately labeled and then certaincellular test colonies are inoculated with a quantity of both thelabeled and unlabeled material after which binding studies are conductedto determine the extent to which the labeled material binds to the cellreceptors. In this way, differences in affinity between materials can beascertained.

Accordingly, a purified quantity of the weight modulator may beradiolabeled and combined, for example, with antibodies or otherinhibitors thereto, after which binding studies would be carried out.Solutions would then be prepared that contain various quantities oflabeled and unlabeled uncombined weight modulator, and cell sampleswould then be inoculated and thereafter incubated. The resulting cellmonolayers are then washed, solubilized and then counted in a gammacounter for a length of time sufficient to yield a standard error of<5%. These data are then subjected to Scatchard analysis after whichobservations and conclusions regarding material activity can be drawn.While the foregoing is exemplary, it illustrates the manner in which areceptor assay may be performed and utilized, in the instance where thecellular binding ability of the assayed material may serve as adistinguishing characteristic. In turn, a receptor assay will beparticularly useful in the identification of the specific receptors tothe present modulators, such as the DB receptor.

A further assay useful and contemplated in accordance with the presentinvention is known as a "cis/trans" assay. Briefly, this assay employstwo genetic constructs, one of which is typically a plasmid thatcontinually expresses a particular receptor of interest when transfectedinto an appropriate cell line, and the second of which is a plasmid thatexpresses a reporter such as luciferase, under the control of areceptor/ligand complex. Thus, for example, if it is desired to evaluatea compound as a ligand for a particular receptor, one of the plasmidswould be a construct that results in expression of the receptor in thechosen cell line, while the second plasmid would possess a promoterlinked to the luciferase gene in which the response element to theparticular receptor is inserted. If the compound under test is anagonist for the receptor, the ligand will complex with the receptor, andthe resulting complex will bind the response element and initiatetranscription of the luciferase gene. The resulting chemilurninescenceis then measured photometrically, and dose response curves are obtainedand compared to those of known ligands. The foregoing protocol isdescribed in detail in U.S. Pat. No. 4,981,784 and PCT InternationalPublication No. WO 88/03168, for which purpose the artisan is referred.

Once a recombinant which expresses the OB receptor gene sequence isidentified, the recombinant OB receptor can be analyzed. This isachieved by assays based on the physical or functional properties of theOB receptor, including radioactive labeling of the receptor followed byanalysis by gel electrophoresis, immunoassay, ligand binding, etc.Furthermore, antibodies to the OB receptor could be generated asdescribed above.

The structure of the OB receptor can be analyzed by various methodsknown in the art. Preferably, the structure of the various domains,particularly the OB binding site, is analyzed. Structural analysis canbe performed by identifying sequence similarity with other knownproteins, particular hormone and protein receptors. The degree ofsimilarity (or homology) can provide a basis for predicting structureand function of the OB receptor, or a domain thereof. In a specificembodiment, sequence comparisons can be performed with sequences foundin GenBank, using, for example, the FASTA and FASTP programs [Pearson etal., Proc. Natl. Acad. Sci. USA, 85:2444-2448 (1988)].

The protein sequence can be further characterized by a hydrophilicityanalysis (e.g., Hopp et al., 1981, supra). A hydrophilicity profile canbe used to identify the hydrophobic and hydrophilic regions of the OBreceptor protein, which may in turn indicate extracytoplasmic, membranebinding, and intracytoplasmic regions.

Secondary structural analysis (e.g., Chou et al., 1974, supra) can alsobe done, to identify regions of the OB receptor that assume specificsecondary structures.

Manipulation, translation, and secondary structure prediction, as wellas open reading frame prediction and plotting, can also be accomplishedusing computer software programs available in the art.

By providing an abundant source of recombinant OB polypeptide, and theopportunity to isolate the OB receptor (i.e., the DB gene product), thepresent invention enables quantitative structural determination of theactive conformation of the OB polypeptide and the OB receptor, ordomains thereof. In particular, enough material is provided for nuclearmagnetic resonance (NMR), infrared (IR), Raman, and ultraviolet (UV),especially circular dichroism (CD), spectroscopic analysis. Inparticular NMR provides very powerful structural analysis of moleculesin solution, which more closely approximates their native environment(Marion et al., 1983, supra; Bar et al., 1985, supra; Kimura et al.,1980, supra). Other methods of structural analysis can also be employed.These include but are not limited to X-ray crystallography (Engstom,1974, supra).

More preferably, co-crystals of OB polypeptide and OB receptor can bestudied. Analysis of co-crystals provides detailed information aboutbinding, which in turn allows for rational design of ligand agonists andantagonists. Computer modeling can also be used, especially inconnection with NMR or X-ray methods [Fletterick et al., eds., ComputerGraphics and Molecular Modeling, in Current Communications in MolecularBiology, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1986)].

Identification and isolation of a gene encoding an OB receptor of theinvention provides for expression of the receptor in quantities greaterthan can be isolated from natural sources, or in indicator cells thatare specially engineered to indicate the activity of a receptorexpressed after transfection or transformation of the cells.Accordingly, in addition to rational design of agonists and antagonistsbased on the structure of the OB polypeptide, the present inventioncontemplates an alternative method for identifying specific ligands ofOB receptor using various screening assays known in the art.

The invention may be better understood by reference to the followingExamples, which are intended to be exemplary of the invention and notlimiting therof.

EXAMPLE SECTION

The following outlines the method used to identify the genetic materialthat is exemplary of the present invention. This endeavor comprises foursequential steps: A) Genetic Mapping, B) Physical Mapping, C) CandidateGene Isolation, and D) Mutation detection. Following confirmation thatthe murine gene in object was isolated (Step D), the homologous humangene was sought, and both the murine and human genes and putativeproteins were characterized. The steps are summarized in greater detail,below.

A. Genetic Mapping

The ob mutation was segregated in genetic crosses, and standard linkageanalysis was used to position the mutation relative to RFLPs(restriction fragment length polymorphisms). These data placed the OBgene in an ˜ 5cM interval on proximal mouse chromosome 6. (ScM is ameasurement of genetic distance corresponding to 5 apparent geneticcrossovers per 100 animals.) A total of 771 informative meioses weregenerated and used in subsequent genetic mapping (Friedman et al., 1991,supra). The genetic loci that were mapped relative to OB were allpreviously published. The two closest RFLPs described were defmed byprobes derived from the carboxypeptidase and met oncogene genes.

The genetic resolution of the experiments described above was inadequateto clone OB, principally because none of the genetic markers were intight linkage. In order to identify the requisite tightly linked RFLPs,additional probes were isolated and the genetic cross was expanded. Amethod known as chromosome microdissection was used to isolate randompieces of DNA from proximal mouse chromosome 6 [Bahary et al., MammalianGenome, 4:511-515 (1993)]. Individual cloned probes were tested fortight linkage to OB. On the basis of these studies one probe, D6Rck13,also termed psd3, was selected for further analysis owing to its geneticproximity to OB.

This probe was used to genotype 835 OB progeny from interspecific andintersubspecific crosses, which indicated that D6Rck13 is nonrecombinantin all 835 animals as reported in Bahary et al. In the course ofphysical mapping, a new polymorphic marker was identified from a cosmidsubclone derived from YAC 53A6. This new marker was positioned betweenD6Rck13 and the OB gene and was used to genotype the additional 771informative meioses from intraspecific intercross and backcross. Asingle animal #167 was identified to bear a recombination crossoverbetween OB and D6Rck39. These studies indicated that D6Rck39/D6RcK13 is˜ 0.06 cM from OB. An additional probe, Pax4, was identified that was0.12 cM proximal to OB. Pax4 was recombinant in two animals; #111 and#420. Pax4 is a pseudogene that was previously mapped to proximal mousechromosome 6 by Gruss and co-workers [Gruss et al., Genomics, 11:424-434(1991)]. On this basis, it was determined that the OB gene resides inthe ˜ 0.2cM interval between Pax4 and D6Rck13. This led to efforts toclone the interposing DNA in an effort to isolate OB.

B. Physical Mapping

The cloning of the DNA in this interval made use of yeast artificialchromosomes (YACs), a relatively new cloning vector that allows thecloning of long stretches of contiguous DNA often more than one millionbase pairs in length.

Yeast artificial chromosomes were isolated using D6Rck13 and Pax4. Thiswas accomplished by preparing purified DNA probes and using them toisolate the corresponding YACs. These YACs (#8, #16, #107 and #24) wereisolated and initially characterized, and on the basis of the resultinganalyses it was concluded that YAC 16 was the YAC that extended furthestdistally, i.e., closest to OB. The key end of YAC #16 was thenrecovered, and it was determined that this end was closer to OB thanPax4. This end was termed 16M(+). This conclusion was reached because itwas shown that this probe was not recombinant in animal #420 (as wasPax4). This end was sequenced and used to develop a PCR assay. This PCRassay was used to screen a YAC library. Four positive clones wereisolated. Subsequent characterization of these YACs by end-rescuing,restriction mapping, pulse field gel electrophoresis, and Southern blotswith the genetic crosses determined that two of these YACs, adu and aad,were critical for subsequent studies. YAC aad is a 550 kB nonchimericYAC which extended furthest distally. Therefore, the distal end of thisYAC, aad(pICL) was used to complete the physical map. YAC adu is a 370kb nonchimeric YAC and its distal end, adu(+), was determined to benonrecombinant in all the OB progeny of the genetic crosses includinganimals #111 and #167, suggesting that the OB gene might reside in thisYAC.

A PCR assay for these two ends, aad(pICL) and adu(+) was developed andused for isolating more YACs and P1 clones to continue physical mapping.The important P1 clones isolated by this effort included 498, 499, 500(isolated using a probe derived from aad(pICL)) and 322, 323 and 324(using a probe from adu(+)).

In the meantime, YACs isolated by D6Rck13 (53A6, 25A8, 25A9, 25A10) werecharacterized. These studies determined that 53A6 extended furthestproximally toward the aad YAC. The size of the gap between 53A6 and aadwas determined to be ˜70 kB. The key end of 53A6, 53(pICL) was then usedto screen three available YAC libraries and a P1 library. A critical P1clone, 325, was isolated. This P1 clone overlapped with the P1 clonesisolated by aad(pICL) as described above, and therefore served to closethe gap between 53(pICL) and aad(pICL). As a result, the whole contig,containing YACs and P1 clones, of ˜2.5 million base pairs in length, andspanning Pax4, 16M(+), adu(+), aad(pICL), 53(pICL), D6Rck39 and D6Rck13,was cloned. By carefully mapping the sites of recombination apparent inanimal #111 and #167, it was concluded that OB was situated in a 400 kBinterval. To provide a working DNA source for isolating the OB gene,about 500 kB covering this nonrecombination region was isolated in atotal of 24 P1 clones. These P1 clones, including 322 and 323, whichlater were found to be useful clones, were used for exon trapping.

The physical map of the portion of the chromosome carrying OB is shownin FIG. 7A. FIG. 7B represents the YAC contig. FIG. 7C represents the P1contig.

C. Isolation of Candidate Genes

The method used to isolate genes in this interval was exon trapping.This method used a commercial vector to identify exon DNA (i.e., codingsequences) by selecting for functional splice acceptor and donorsequences in genomic DNA introduced into a test construct. The DNA fromthese P1 clones were grown and subcloned into the exon trapping vector.These clones were short inserts cloned into a Bluescript vector. Eachclone was PCR amplified with PCR primers corresponding to plasmidsequences that flanked the insert. The PCR amplification was performeddirectly on the bacteria that carried the plasmid. The reactions wereset up using a Biomek robot. The PCR products were electrophoresed on a1% agarose gel in TBE buffer that contained ethidium bromide. The exontrapping technique was modified to eliminate contaminating E. coli DNAfrom the P1 clones, and to screen out the abundant artifactual exons,which exceeded 80-90% of the putative exons trapped. The exon trappingvector includes HIV sequences; a short segment of these vector sequencescorresponds to this artifact.

The exon trapping experiment was performed using various P1 clones. Exontrapping products were then amplified by PCR, selected, and sequenced.Sequences of putative "exons" were compared with those in Genbank usingthe Blast computer program. About fifteen exons were selected forfurther examination by RT-PCR, Northern analysis, and zoo blot for thepresence of corresponding RNA or conserved sequences. Seven of thefifteen putative exons, 325-2, 323-9, 322-5, D1-F7, 1H3, and 2G7, werefound to encode an RNA transcript. 325-2 is a testis specific gene;323-8 and 323-9 are likely two exons from the same gene expressed mainlyin brain and kidney. IH3 and 322-5 represent two low level braintranscripts. D1-F7 is an exon from a previously cloned gene, inosinemonophosphate dehydrogenase (IMPDH), which has ubiquitous expressionpattern. None of these genes appeared to encode OB. 2G7, which is the OBexon, is discussed further below.

After three unsuccessful efforts to exon trap the OB gene, anotherattempt was made by pooling DNA from all the P1s from the critical OBregion. These included P1s: 258, 259, 322, 323, 324, 325, 498, 499, 500,653, 654 and others. Thereafter P1s 258, 260, 322, 498 and 499 weresubcloned into the exon trapping vector, and subsequently several plateswere prepared with bacterial clones, each of which carried a putativeexon. Approximately 192 clones representing putative OB candidates wereobtained. As noted above, a consistent artifact such that many of theisolates contained two trapped exons derived from the vector wasobserved. Thus, clones were identified both by their size and by thefact that hybridization of DNA probes corresponding to this artifacthybridized to the corresponding bands on a Southern blot of the gel. Inthis way, 185 out of 192 clones were excluded from further evaluation.Exclusion of the artifacts on the basis of size alone was not possible,as this could have, in the end, led to exclusion of the exoncorresponding to OB.

Thus, of the 192 exons, a total of seven exons were selected for furtherstudy. Templates for sequencing the seven exons were prepared, andsequencing was performed. The sequences for the seven exons wereanalyzed and it was found that four were identical and one was anapparent artifact. In particular, clone 1D12 contained the "HIVsequence," i.e., the artifact band. This left three exons for furtheranalysis: 1F1, 2G7 and 1H3. 1F1 was eliminated because it mapped outsidethe critical region. PCR primers for both 1H3 and 2G7 were selected andsynthesized.

The sequence of the exon on 2G7 was determined, and is shown in FIG. 10(SEQ ID NO:7). PCR primers for 2G7 were selected and synthesized. Theportions of the sequence corresponding to the PCR primers areunderlined. The primers used were:

     5'CCA GGG CAG GAA AAT GTG                                                                           (Tm = 60.0° C.)                                 (SEQ ID NO:8)                                                                 3'CAT CCT GGA CTT TCT GGA TAG G                                                                        (Tm = 60.0° C.)                               (SEQ ID NO:9)                                                             

These primers amplified genome DNA with PCR conditions as follows: 25-30cycles at 55° C. annealing for 2', 72° C. extension for 2', 94° C.denaturation for 1' in standard PCR buffer. These primers were also usedto generate a labeled probe by including ³² P-dCTP in the PCR reactionwith a corresponding reduction in the amount of cold dCTP.

A RT-PCR was performed on a variety of tissue RNAs and it was concludedthat 2G7 was expressed exclusively in white fat among the tissuesexamined (FIG. 11A). Thereafter, ³² P-labeled 2G7 was hybridized to aNorthern blot of tissue RNAs (FIG. 11B) and showed that its RNA wasexpressed at high level in fat tissue but was either not expressed orexpressed at very low levels in all other tissues (where the signals maybe the result of fat contaminating the tissue preparations). Ten μg oftotal RNA from each of the tissues listed was electrophoresed on anagarose gel with formaldehyde. The probe was hybridized to the blot at65° C. in a standard hybridization buffer, Rapid Hybe (Amersham). Thesize of the RNA was approximately 4.9 kB. At this point 2G7 wasconsidered to be a viable candidate gene for OB and was analyzedfurther.

D. Mutation Detection

In order to confirm that 2G7 encoded the OB gene, it was necessary todemonstrate differences in the levels of RNA expression of the DNAsequence of this gene in mutant as compared to wild-type animals. Twoseparate mutations of the OB gene are available for study, C57BL/6Job/ob (1J) and Ckc/Smj oblob (2J). These will hereinafter be referred toas 1J and 2J, respectively. (Informal nomenclature is used to refer tothe mouse strains studied. Throughout this specification and in thedrawings, it will be understood that C57BL/6J refers to C57BL/6J +/+;CKC/smj refers to SM/Ckc-+^(Dac) -+/+; CKC/smj ob/ob refers toSM/Ckc-+^(Dac) -ob^(2J) /ob^(2J)). RNA was prepared from fat tissue thathad been isolated from 1J, 2J, and control animals. Total RNA for eachsample was treated with DNase and then reverse transcribed usingoligo-dT as a primer and reverse transcriptase. The resultingsingle-stranded cDNA was then PCR amplified either with the 2G7 primers(conditions shown above) for the lower band or with commerciallyavailable actin primers for the upper band. The RT-PCR products were runon a 1% agarose TBE gel that was stained with ethidium bromide (FIG.12A). Using RT-PCR it was found that while 2G7 mRNA was expressed in 1Jand all the other control mice, it was completely missing in 2J mouse.No signal was detected after 30 cycles of amplification. This experimentprovided direct evidence that 2G7 corresponded to an exon from the OBgene.

Since the 2J mutation is relatively recent and is maintained as acoisogenic strain, this result was the first available evidence thatindicated that 2G7 is an exon from the OB gene. The mutation is likelylocated in the promoter region which leads to total abortion of mRNAsynthesis. The presence of a signal in 1J mouse in this RT-PCRexperiment suggested that 1J might carry a point mutation which does notresult in a gross change in size of the RNA sample. In addition, 2G7mRNA was absent, when tested by RT-PCR, from four additional 2J animals.

This result was confirmed on a Northern blot (FIG. 12B). Fat cell RNAwas prepared from each of the strains (C57B1/6J, 1J, CKC/smj, and 2J).Ten μg of these RNAs were run out and blotted. The blot was probed withthe 2G7 probe that was PCR-labeled, by amplification of the material,i.e., band, in FIG. 11 using ³² P-dCTP in the PCR reaction. Actin is acontrol for the amount of RNA loaded. The actin signal is fairly similarin all of the samples. The OB signal is absent in brain because the mRNAis specific to fat cells.

The results of the Northern analysis confirm that 2G7 specific RNA isabsent in 2J mice. The ob RNA is absent in the CKC/smj ob/ob micebecause in this obese mutant strain the gene is disrupted such that noRNA is made. In addition, the level of 2G7 RNA was increased ˜10-20 foldin 1J as well as db/db fat. These results are compatible with thehypothesis that OB either encodes circulating hormone or is responsiblefor the generation of a signal from fat cells that modulates bodyweight. These results supported the conclusion that 2G7 is the OB geneand predicted that 1J mice have a point mutation, probably a nonsensemutation leading to a premature translation termination.

These Northern results have been replicated using fat cell RNApreparations from four different 2J animals (FIG. 13). In this assay,ap2 is a fat-specific transcript that was used as a control much thesame as actin in FIG. 12B. There is no significance to the varyingdensity of the ap2 band. ap2 was labeled by designing PCR primers formthe published ap2 sequence. The RT-PCR products of fat cell RNA werethen relabeled using the same protocol for PCR labeling. This analysisdemonstrates the presence of OB mRNA in normal homozygous orheterozygous animals, and its absence from 2J mutant animals.

The mutation has been identified in 1J mice. The mutation is a C to Tbase change that results in a change of an arginine to an apparentpremature stop codon at amino acid 108, and in all likelihood accountsfor the 1J mutation (FIG. 14) despite high level expression of the obmRNA (see FIGS. 12 and 13, C57BL/6J ob/ob lanes).

More recently, Southern blots have been used to conclude that the 2Jmutation is the result of a detectable DNA change at the 5' end of OBthat appears to completely abolish RNA expression. The exact nature ofthis possible rearrangement remains to be determined.

A genomic Southern blot of DNA from the CKC/smj (SM/Ckc-+^(Dac)) andC57BL/6J mice using four different restriction endonucleases wasperformed in order to determine whether the mutant ob yielded a uniquefragment pattern (FIG. 15A). Approximately 10 μg of DNA (derived fromgenomic DNA prepared from liver, kidney, or spleen) was digested withthe restriction enzyme indicated. The DNA was then electrophoresed in a1% agarose TBE gel. The DNA was transferred to an imobilon membrane andhybridized to the PCR-labeled 2G7 probe. The key band is the uppermostband in the BglII digest for the CKC/smj ob/ob (SM/Ckc-+^(DAC) ob^(2J)/ob^(2J)) DNA. This band is of higher molecular weight than in the otherstrain, indicating a mutation in this strain. FIG. 15B is a Southernblot of a BglII digest of genomic DNA from the progeny of an ob^(2J) /+xob^(2J) /+cross. Some of the DNAs have only the upper band, some onlythe lower band, and some have both bands. The animals with only theupper band are allo-obese, i.e., ob^(2J) /ob^(2J). These data show thatthe polymorphism (i.e., mutation) shown in FIG. 15A segregates in agenetic sense.

Example 1: cDNA Cloning and Sequence Determination of OB

Using the labeled 2G7 PCR probe, a total of fifty mouse cDNA clones froma murine fat cell λgt11 cDNA library (Clonetech 5'-STRETCH cDNA fromtesticular fat pads of Swiss mice, #ML3005b), and thirty crosshybridizing human cDNA clones from a human fat cell λgt10 cDNA library(Clonetech 5'-STRETCH cDNA from abdomen #HL1108a) were isolated. Libraryscreening was performed using the plaque lift procedure. The filtersfrom the plaque lift were denatured using the autoclave method. Thefilters were hybridized in duplicate with the PCR-labeled 2G7 probe(Rapid Hybe buffer, 65° C., overnight). After a 2-4 hourprehybridization, the filters were washed in 2x SSC, 2% SDS, twice for30 minutes at 65° C. and exposed to x-ray film. Duplicate positives wereplaque purified. Plaque purified phage were PCR-amplified usingcommercially available vector primers, e.g., λgt10 and λgt11. Theresulting PCR products corresponded to the cDNA insert for each phagewith a small amount of vector sequence at either end. The bands were gelpurified and sequenced using the ABI automated sequencer and the vectorprimers to probe the DNA polymerase.

The raw sequencing data were then manually examined base by base tocorrect mishearing from the computer program. As the correct sequencebecame available, the downstream primers were synthesized and used tocontinue sequencing. Such experiments were repeated until each availablecDNA clone was sequenced and synthesized into a contig. To date, ˜3000base pairs from the 5' end of the mRNA has been compiled. One of thecDNA clones extended to the 5' end of the mRNA since its sequence wasidentical to that of the 5' RACE product of fat tissue RNA (data notshown).

The sequence data revealed that there is a 167 amino acid open readingframe (FIG. 1). A Kozak translation initiation consensus sequence waspresent with an adenosine residue three bases upstream of the ATG. Twoclasses of cDNA were found differing by inclusion or exclusion of asingle glutamine codon. This residue is found in a position immediately3' to the splice acceptor of the 2G7 exon. Since the CAG codon ofglutamine includes a possible AG splice acceptor sequence, it appearsthat there is slippage at the splice acceptor site with an apparent 3base pairs deletion in a subset of the cDNA, as shown below.

               gln ser  val                                                               ag CAG TCG GTA                                                                         (with              (SEQ ID NO:17)                                       ↑                                                                             glutamine)                                                   (splice acceptor site)                                                                       ser  val                                                               ag CAG TCG GTA                                                                         (without                                                                   ↑                                                                         glutamine)                                                         (splice acceptor site)                                               

The "ag" in the sequences above corresponds to the assumed intronsequence upstream of the glutamine codon, and AG is the putativealternative splice site. This glutamine residue is located in a highlyconserved region of the molecule and its importance for biologicalactivity is as yet unknown.

A putative N-terminal signal sequence was detected, the signal cleavagesite of which is predicted to be carboxy-terminal to the alanine residueat amino acid position 21. This putative signal sequence was confirmedby application of a computer algorithm to the method of von Heijne,Nucl. Acids Res., 14:4683 (1986). Using this technique, the mostprobable signal sequence was identified in the polypeptide coding regioncorresponding to amino acids 1-23, having the sequence:

    MCWRPLCRFLWLWSYLSYVQA ↑ VP (SEQ ID NO:10)

in which the arrow indicates the putative signal sequence cleavage site.The rest of the amino acid sequence was largely hydrophilic and did nothave any notable structural motifs or membrane spanning domains otherthan the N-terminal signal sequence. Specifically, we did not findconsensus sequences for N-linked glycosylation or dibasic amino acidsequences indicative of protein cleavage in the predicted processedprotein (Sabatini et al., The Metabolic Basis of Inherited Disease, pp.177-223, C. V. Scriver et al. eds., McGraw-Hill, N.Y.). Data basesearches using Blast and Block programs did not identify any homologoussequences.

Human fat tissue RNA was analyzed on Northern blots, RNA species of asimilar size to the mouse OB gene was detected. Sequencing and analysisof cDNA clones revealed that human OB also encodes a 167 amino acidpolypeptide (FIG. 2A and B and FIG. 3). Two classes of cDNA, with orwithout three base pair deletions, were found in human as well (FIG. 6).The mouse and human OB genes were highly homologous in the predictedcoding region, but had only 30% homology in the available 3' and 5'untranslated regions. An N-terminal signal sequence was also present inthe human OB polypeptide. Comparison of the human and mouse OBpolypeptide sequences showed that the two molecules share an overall 83%identity at the amino acid level (FIG. 4). The N-termini of the matureproteins from both species share even higher homology, with only sixconservative and three nonconservative amino acid substitutions amongthe N-terminal 100 amino acid residues.

Genomic DNA was isolated from mouse, rat, rabbit, vole, cat, cow, sheep,pig, human, chicken, eel, and Drosophila, and restriction digested withEcoRI. The digests were electrophoresed on 1% agarose TBE gel. DNA wasthen transferred to an immobilon membrane and probed with thePCR-labeled 2G7 probe. The filter was hybridized at 65° C. and washedwith 2x SSC, 0.2% SDS at 65° C. twice for twenty minutes each wash,i.e., there were two buffer changes. These data indicate that OB isconserved among vertebrates (FIG. 16). Note in this regard that there isa 2+signal in eel DNA; eel is a fish.

In summary, available evidence suggests that body weight and adiposityare physiologically controlled. Seven years ago efforts began toidentify two of the key components of this system: the OB and DB genes.As shown in this example, the OB gene has now been identified as a fatspecific gene that plays a key role in regulating body weight. Theproduct of this gene, which is most probably a secreted hormone, willhave important implications for the diagnosis and treatment ofnutritional disorders in man and non-human animals.

Example 2: Expression of OB In Bacteria

Both murine and human cDNAs encoding OB have been cloned into a pET-15bexpression vector (Novagen). This vector contains a T7 promoter inconjunction with a lac operator, and expresses a fusion proteincontaining a histidine tag (His-tag) and a thrombin cleavage siteimmediately upstream of the coding sequence insertion site (FIG. 17)(SEQ ID NOS:11 and 12).

The mouse and human cDNAs were modified such that the alanine at the endof the signal sequence was turned into an NdeI site, as was a separatesequence in the 3' region. Insertion of the NdeI site was accomplishedusing PCR with novel primers:

Mnde-5' (murine five prime primer):

CTTATGTTCA TATGGTGCCG ATCCAGAAAG TC (SEQ ID NO:13)

Mnde-3' (murine three prime primer):

TCCCTCTACA TATGTCTTGG GAGCCTGGTG GC (SEQ ID NO:14)

Hnde-5' (human five prime primer):

TCTATGTCCA TATGGTGCCG ATCCAAAAAG TC (SEQ ID NO:15)

Hnde-3' (human three prime primer):

TTCCTTCCCA TATGGTACTC CTTGCAGGAA GA (SEQ ID NO:16)

The primers contain a 6-base pair mismatch in the middle that introducesNdeI restriction sites at each end of the PCR fragment. Phage carryingeither the mouse or human cDNA were PCR amplified using those primers.The PCR product was digested with NdeI and gel purified on a 1% lowmelting point agarose gel. The gel purified bands were subcloned intothe pET vector. The resulting plasmids were sequenced to ensure thatmutations were not introduced during the PCR amplification step ofcloning. Constructs for the human and murine cDNA that encode and thatlacks glutanine 49 have been prepared. In particular, pET 15b constructscontaining either the human or the mouse OB coding sequence, minussignal sequence and fused to a His-tag, have been made using a PCRcloning method. The constructs have been sequenced to ensure no sequenceerrors were introduced into the coding region of the OB gene during thePCR amplification step.

Two resultant plasmid constructs, pETM9 and pETH14, were selected totransform a bacterial expression host. Upon induction with 1 mM IPTGunder optimal conditions, the transformed bacteria were able to produce100-300 μg/ml of the OB fusion. The majority of the OB fusion proteinwas found in the inclusion body. After solubilization with 6Mguanidine-HCl or urea, the fusion protein was purified through aHis-binding (Ni-chelation) resin column. The conditions for columnpurification of the OB fusion protein (including binding, washing, andeluting) were established experimentally. The OB fusion protein binds tothe resin at 5 mM imidazole/6M guanidine-HCl and stays bound at up to 20mM imidazole/6M guanidine-HCl. The protein can be eluted from the resinat 60 mM imidazole/6M guanidine (FIG. 18A,B). Both the purified humanand mouse OB fusion proteins were further dialyzed in PBS to removeguanidine-HCl from the preparation, then used to raise polyclonalantibodies.

In order to test the biological activity of the fusion protein products,the refolding conditions for the purified protein were tested anddeveloped. This involves initial dialysis of the fusion protein in a 1 Mguanidine solution, followed by dilution with a 0.4 M arginine solution.The His-tag was removed from the fusion proteins before assaying forbiological function. The tag removal was achieved by treating the fusionprotein with thrombin from human placenta.

In addition, human and mouse OB gene coding sequences minus the signalsequence are each being inserted into a pET 12c vector using PCR cloningmethod. These constructs can direct the synthesized OB fusion proteinsinto the periplasmic space of the bacterial host cell. The OB fusionprotein recovered from the periplasmic space may only need a simple gelfiltration to be purified from other host proteins and will not bedenatured during such a process.

Example 3: Preparation of Antibodies to the OB Polypeptide

In addition to use of the recombinant protein to generate polyclonalantibodies, a set of four peptide sequences from the deduced murine OBsequence were identified using immunogenicity plot software (GCGPackage). The four carboxyl terminal peptide fragments are:

(SEQ ID NO:18):

Val--Pro--Ile--Gln--Lys--Val--Gln--Asp--Asp--Thr--Lys--Thr--Leu--Ile--Lys--Thr

(SEQ ID NO:19):

Leu--His--Pro--Ile--Leu--Ser--Leu--Ser--Lys--Met--Asp--Gln--Thr--Leu--Ala

(SEQ ID NO:20):

Ser--Lys--Ser--Cys--Ser--Leu--Pro--Gln--Thr--Ser--Gly--Leu--Gln--Lys--Pro--Glu--Ser--Leu--Asp

(SEQ ID NO:21):

Ser--Arg--Leu--Gln--Gly--Ser--Leu--Gln--Asp--Ile--Leu--Gln--Gln--Leu--Asp--Val--Ser--Pro--Glu--Cys

These peptides were conjugated to KLH, and the peptide-KLH conjugateswere used to immunize rabbits using standard techniques. Polyclonalantisera specific for each peptide is recovered from the rabbits.

Example 4: In Vitro Translocation of an OB Polypeptide

In order to confirm the presence of a functional signal sequence, ahuman cDNA that included the entire open reading frame was subclonedinto the pGEM vector. Only the human cDNA was used in this experimentbecause suitable mouse subclones were not recovered. Positive strandhuman ob RNA was transcribed using Sp6 polymerase and used in an invitro translation reaction with and without canine pancreatic microsomalmembranes. The primary translation product migrated with an apparentmolecular weight of ˜ 18 kD, which is consistent with that predicted bythe cDNA sequence. Inclusion of the microsomal membranes in the reactioninhibited the overall efficiency of translation ˜ 5-fold. Nevertheless,approximately 50-70% of the OB primary translation product was truncatedby approximately 2 kD in the presence of the membrane preparation,suggesting that the signal sequence is functional (FIG. 19A). The sizeof the primary translation product of interleukin-1α RNA, which does notencode a signal sequence, was unchanged when microsomal membranes wereincluded in the reaction. In order to confirm that translocation of theOB protein had taken place, the in vitro translation products weretreated with Proteinase-K. Protease treatment resulted in the completeproteolysis of the 18 kD primary translation product while the 16 kDprocessed form was unaffected by the enzyme treatment, indicating thatit had translocated into the lumen of the microsomes (FIG. 19B). Thesedata are compatible with the hypothesis that OB is a secreted molecule.

After signal sequence cleavage, two cysteine residues would remainwithin the predicted protein raising the possibility that the moleculecontains a disulfide bond characteristic of other secreted polypeptides[Shen et al., Science, 224:168-171 (1984)].

Example 5: Characterization of the OB Gene

To establish the relationship between obesity and genetic alterations inthe OB gene in humans, the sequence of the human OB gene was determined(FIG. 20A through C) (SEQ ID NOS:22 and 24). Specific primers from thehuman coding sequence were used to screen a human P1 library. Threedifferent P1 clones were obtained, grown up, and PCR amplified usingprimers flanking the splicing site between the first and second codingexons. The entire intron region, around 2 kb, was amplified andpartially sequenced (see FIG. 20A; and as indicated in SEQ ID NOS:22 and24).

The gene structure of both the murine and human genes was characterizedusing PCR assays and other standard techniques. The mouse OB gene wasfound to consist of three exons, the second and third of which accountfor the coding sequence (FIG. 20D). The coding region of the human OBgene shares the same structure; however, the human gene lacks a 5' exonand intron (FIG. 20E).

Two sets of primers generated from the intronic sequences of the humangene have been prepared (FIG. 20A through C). The sequences of theprimers follows (F and R refer to forward and reverse, respectively):

HOB 1gF 5'-CCCAAGAAGCCCATCCTG-3' (SEQ ID NO:29)

HOB 2gR 5'-GACTATCTGGGTCCAGTGCC-3' (SEQ ID NO:30)

HOB 2gF 5'-CCACATGCTGAGCACTTGTT-3' (SEQ ID NO:31)

HOB 2gR 5'-CTTCAATCCTGGAGATACCTGG-3' (SEQ ID NO:32)

DNA samples have been obtained from various sources, and these sets ofprimers are being used to amplify human genomic DNA from severely obesepeople. The PCR products were run on a low melting point agarose gel,and the bands were cut out and digested with agarase. The sequences wereobtained using the ABI 373A DNA sequencer and Taq dideoxy terminator kit(ABI, Perkin-Elmer). One point mutation in an ob gene from a patientsample has been detected to date. This mutation is in the first exon anddoes not change the amino acid sequence. Preliminary data indicate thatan insertion sequence may be present in the first exon of anotherpatient.

A different automated sequencing method using Sequenase instead of TaqDNA polymerase may be employed to yield more easily readable sequencesfor mutation detection.

Example 6: Expression of OB in Yeast

Following the positional cloning of OB, it became important to uncoverthe physiological mechanism by which the OB protein reduces food intakeand body weight. The first step in this direction was to recombinantlyproduce a functional protein using an expression system. In addition tothe successful bacterial expression system, a yeast expression systemwas also selected. Yeast expression has several attractive features forexpressing OB. The most important is that biologically active eukaryoticproteins are more likely to be produced. The OB polypeptide is secretedby mammalian cells. Protein secretion is very similar for alleukaryotes, which means that the yeast secretory apparatus is much moresimilar to the mammalian secretory pathway than bacterial secretorypathways would be. In particular, protein modifications of OB seen inmammalian cells would likely also be seen in the expression through theyeast secretory system. In addition, protein folding is carried out inpassage through the secretory apparatus and thus, delivering OB throughthe yeast secretory apparatus is likely to give a properly foldedprotein with native biological activity. This is significant for OBbecause the two cysteine residues may form a disulfide bridge. Incontrast to secretory pathways, the reducing environment of the cellcytoplasm prevents formation of disulfide bridges; and therefore it isessential that OB pass through the secretory pathway in order for thisdisulfide bond to form in vivo. Other advantages have to do with theease and quickness of manipulating yeast, the availability of vectorsand strains, and the vast experience in yeast recombinant technology.

A Pichia pastoris expression system was chosen for four reasons: (1) ithas higher levels of heterologous protein expression than other yeastsystems such as S. cerevisiae; (2) protein glycosylation is more similarto the mammalian system in P. pastoris than in S. cerevisiae (althoughglycosylation sites were not detected in ob using a computer search,there still remained the possibility of glycosylation at unrecognizedsites); (3) P. pastoris secretes very few proteins natively, and thus itis generally straightforward to purify the expressed foreign protein;and (4) the vectors and yeast strains are commercially available (fromInvitrogen). Two strategies for generating yeast expression vectors areshown in FIGS. 21 and 22.

The vector chosen was pPIC.9. This vector contains a cloning site justdownstream of the α-mating factor prepro coding sequence which directsthe protein encoded by the gene cloned into the cloning site to besecreted by the secretory pathway. The other important feature of thevector is a HIS4 gene that allows selection for uptake of the vectorusing a yeast auxotrophic strain grown on histidine-deficient mediafollowing transformation of the yeast with the vector. The cloningstrategy was as follows: PCR amplify OB cDNA using a 5' primer thatcontains at its 3' end, sequence complementary to the sequence of OBjust following the predicted leader peptide cleavage site, and at itsmost 5' end, a sequence complementary to the 3' end of the α-matingfactor sequence of the vector. The 5' primer also contains an XhoI site.The 3' primer was designed to have at its 3' end a sequencecomplementary to the last few amino acids of OB and an EcoRI site at its5' end. Following PCR amplification, the PCR product was digested withXhoI and EcoRI and cloned into similarly digested pPIC.9. Following thecloning of both the mouse and human OB cDNAs, each with and without theglutamine at codon 49, individual clones were isolated for all fourconstructs and sequenced to verify that the constructs were cloned inthe correct orientation, and frame, and contained no mutations from thePCR amplification step. Following identification of clones with thecorrect sequence, these were transformed into P. pastoris strain GS115,a histidine auxotroph.

For the two mouse OB constructs, transformed yeast clones were screenedfor protein expression. As evidence that the transformed yeast containOB, a DNA dot-blot assay and a colony hybridization assay were donewhich both showed OB sequence within the transformed yeast, but notwithin the untransformed yeast. Furthermore, the transformed yeast nowsecreted a 16 kDa protein into the culture media, whereas theuntransformed yeast does not secrete a protein of this size (FIG. 23A).This is the predicted size of OB. Individual clones for both mouseconstructs have been identified that are high expressors for OB, andcurrently a purification strategy is being developed to purify OB tohomogeneity. One strategy has been to purify OB on a cation exchangecolumn (FIG. 23B); preliminary data suggest that a strong cationexchanger may be useful. However, after cation exchange chromatography,the putative OB product is lost. This indicates the presence of aprotease in the sample.

One strategy to overcome this problem is to prepare OB-His-tag fusionsfor expression in yeast (FIG. 22). Further evaluation has demonstratedthat OB without a His-tag associates tightly with a Ni-chelation column.Purification of the OB polypeptide by Ni-chelation, followed by gelfiltration, yielded a product of sufficient purity for mass spectralanalysis. Mass spec. confirms the molecular weight of the expressedprotein is identical to the expected molecular weight, which stronglyconfirms that OB has been successfully expressed in Pichia.

However, the Ni-chelation/gel filtration purification protocol does notyield an OB polypeptide in sufficiently pure form. Additional smallmolecules are present. It does appear that the proteolytic activityelutes from the Ni-chelation column in the void volume. Accordingly, athree-step purification process is planned: Ni-chelation, followed bycation exchange (which eliminates the small molecule contaminants),followed by gel filtration.

Estimating expression level by Coomassie blue staining of SDS-PAGE gelsreveals approximately 10 mg/l when yeast are grown in shaker flasks.These levels are expected to increase in fermentation vessels, and weare about to initiate fermentation with the hopes of obtaining largerquantities of protein. Regarding the human OB constructs, transformedyeast clones containing high copy numbers of the OB gene have beenidentified, and these are expected to express OB protein. As antibodiesare developed, these will be used to confirm the identity of thesecreted 16 kDa protein.

Example 7: High Level Expression of an OB Fusion Peptide in Bacteria

Preparation offreezer stocks:

To each of the two 4 ml aliquots of sterilized M9ZB media without thecarbon source, 40 μl stock dextrose (0.4 g/ml, filter sterilized) 10 μlampicillin stock (200 mg/ml, and 5 μl chloramphenicol stock (34 mg/ml,in ethanol) were added. A single colony each of E. coli with clonedmouse and human OB1 DNA in a Novagen pET-14b vector was used toinoculate these. The tubes were incubated at 37° C. overnight.

0.5 ml of the overnight cultures were used to inoculate 50 ml M9ZB mediawith dextrose, ampicillin and chloramphenicol. These were incubated at30° C. and the absorbance at 600 mn (A₆₀₀) was monitored periodically.At A₆₀₀ of about 1-1.2, 175 μl aliquots of the culture were mixed with25 μl 60% glycerol in 2 ml eppendorf tubes, flash frozen in liquidnitrogen and stored at -80° C.

Culture growth:

50 ml M9ZB media with 0.5 ml 40% dextrose, 125 μl ampicillin stock and50 μl chloramphenicol stock was inoculated with 1 ml freezer stock andincubated at 30° C. At A₆₀₀ of 1-1.2, 10 ml of this culture was used toinoculate each of four 2 L flasks with 500 ml M9ZB media with dextrose,ampicillin and chloramphenicol. These were incubated at 30° C. untilinduction at A₆₀₀ of about 1-1.2 with a final concentration of 0.5 mMIPTG. The cultures were incubated overnight. The cells were harvested bycentrifugation at 4000 rpm for 20 minutes. This expression system yielda recombinant OB polypeptide as a fairly high percentage of totalprotein; on the order of gram/liter of E. coli.

Cell lysis and resuspension of inclusion bodies:

Cell paste was resuspended in a minimal volume of 20 mM HEPES, pH 7.2,10% glycerol, 0.1 M KCI, 5 mM MgCl₂, 1% aprotinin, 1 mM PMSF, 5 μg/mlleupeptin and 50 μg/ml DNase I. The suspension was freeze thawed threetimes using liquid nitrogen and lukewarm water. Lysed cells werecentrifuged at 18000 rpm for 30 minutes and resuspended in 20 mM HEPES,pH 7.5, 0.1 M NaCl. The suspension was sonicated and Triton X100 wasadded to a final concentration of 2%. This was centrifuged for 15minutes at 18000 rpm. After two more such cycles, three cycles of Tritonfree washes were given. Finally the pellet was dissolved in 6 Mguanidine-HCl (GdHCl), 20 mM HEPES, pH 7.5 by sonication followed bycentrifugation. The supernatant was used for further purification.

The OB protein was purified in the unfolded state by immobilized metalion affinity chromatography (IMAC). The solution was applied to a 40 mlcolumn of Pharmacia chelating fast flow sepharose charged by 5 columnvolumes of 50 mM NiSO₄ and equilibrated in 6 M GdHCl, 20 mM HEPES, pH7.5. The column was washed with 6 M GdHCl, 30 mM imidazole, 20 mM HEPES,pH 7.5. Finally, the protein was eluted with the same buffer containing0.2 M imidazole. Unfolded protein in 6 M GdHCl was stored at 4° C. afteradding sodium acetate (NaAc) to 10 mM and adjusting the pH to about 4.5with acetic acid.

Refolding and the purification of the protein:

6 M GdHCl solution containing 100 mg protein was treated with 67 μl 1 Mdithiothreitol (DTT) and diluted to about 67 ml with 6 M GdHCl, 10 mMNaAc, pH 4.5. It was left stirring at room temperature for about anhour. It was then diluted into 4 L of 20% glycerol, 2.5 mM CaCl₂, 20 mMTris, pH 8.4 buffer with stirring. After proper mixing, the solution wasleft at room temperature for about 8 hours without further stirring.Then 2000 units of purified bovine thrombin (from thrombostat, aParke-Davis product) was added, and the solution was left with gentlestirring. After 2.5 hours it was redosed with 2000 units of thrombin andthe cleavage of the histidine-tag was continued for 3 more hours. Thethrombin cleavage was arrested by adding PMSF to a fmal concentration of0.1 mM. The solution was filtered and stored at 4° C.

The cleaved protein was further purified on the same IMAC column asabove, equilibrated in 1 M KCl, 20% glycerol, 20 mM HEPES, pH 8.4buffer. After loading the protein solution, it was washed with the samebuffer and the cleaved protein was eluted with 1 M KCl, 20% glycerol, 40mM imidazole, 20 mM HEPES, pH 8.4. Uncleaved protein eluted at 0.2 Mimidazole.

Purified cleaved protein was concentrated, treated with 50-100 mM EDTA,10 mM potassium ferricyanide (to complete any incomplete oxidation) andgel filtered on a sephadex 75 16/60 column. Yields using this procedureapproached 50% of the starting peptide.

Once purified, the expressed protein has been characterized by severalmethods. Physical characterization includes dynamic light-scattering todetermine homogeneity of structure and is used as a measure of properfolding. Light scattering data indicate that the human OB polypeptide isexpressed predominantly or exclusively as a monomer, while the murine OBpolypeptide can be found as a dimer as well as a monomer.

Assays with Ellman's reagent and mass spectroscopic analysis confirmthat the cyteine residues form a disulfide bond in the protein. Thisoxidized form of the polypeptide was administered to mice, as describedinfra, and demonstrated biological activity.

Circular dichroism has been used to roughly determine the structuralgeometry of the protein. CD spectra in a physiological buffer (pH about8, approximately physiological ionic strength) indicate that the humanOB polypeptide has about 60% α-helical structure and about 40% randomcoil structure. The murine OB polypeptide was found to have about 50%α-helix and 50% random coil by CD spectroscopy.

Limited proteolysis, followed by mass spectrometry (Cohen et al., 1995,supra) has been employed to identify portions of the OB polypeptide thatare accessible to proteolysis. This analysis has demonstrated thepresence of a flexible loop structure of amino acid residues 54 to 60(as depicted in FIG. 4). It is likely that this flexible loop connectstwo domains of defined 2° structure, e.g., α-helix.

Importantly, as shown in the following Examples, bioactivity of thepurified protein was assayed by administering the protein to both leanand obese rodents via an osmotic pump (e.g., an ALZET osmotic pump fromAlza Corporation, Palo Alto, Calif.) or by daily bolus dose i.p. over atleast a two-week period; and effects on feeding behavior and body weightwere observed.

Example 8: Weight Reducing Effects of the OB Polypeptide (Leptin)

The gene product of the mouse OB locus plays an important role inregulating body weight. The present Example establishes that the OBprotein circulates in mouse, rat and human plasma. The circulating formin all three species has an identical molecular weight by SDS-PAGE tothe deduced polypeptide sequence without the signal sequence, suggestingthat, in vivo, the protein is not processed after cleavage of the signalsequence. The OB protein was absent in plasma from C57/B16J ob/ob miceand present at ten-fold higher concentrations in plasma of db/db miceand twenty-fold higher levels in plasma of fa/fa rats relative tocontrols. It is suggested that db/db and fa/fa obese animal mutants areresistant to the effects of OB. There were seven-fold differences inplasma levels of the OB protein within a group of six lean humansubjects. Daily injections of the recombinant mouse OB proteindramatically reduced body mass in ob/ob mice, had significant effects onbody weight of wild-type mice but had no effect on db/db mice. Thesedata suggest that the gene product of the OB locus serves an endocrinefunction to regulate body weight.

Materials and Methods

Rabbits were immunized with recombinant protein in Freund's adjuvant(HRP, Inc.). Inmunopurified anti-mouse OB antibodies were prepared bypassage of antiserum over a sepharose 4B column conjugated to therecombinant protein as described [Harlow et al., Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1988)]. Immunoprecipitation of mouse plasma was carriedout as follows: 0.5 ml of plasma from mouse, rat and human containingapproximately 2.5 mM EDTA was pre-cleared with unconjugated sepharose-4Bat room temperature with rocking for 2 hours. The sepharose was removedby spinning and 50 ml of a 50% slurry of antibody-conjugated sepharosecontaining affinity-purified antibody at a concentration of 1 mg/ml ofpacked sepharose was added. One-half ml of 2x RIPA buffer was added togive final binding conditions as follows: 50 mM Tris-HCl, pH 7.5, 100 mMNaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate and 0.025% sodiumazide. The reaction was carried out overnight at 4° C. with rocking. Theantibody-conjugated sepharose was washed 8 times using RIPA buffer,followed by rinsing three times with PBS, and run on a 15% SDS-PAGE. Theproteins were transferred to nitrocellulose and Western blotted with abiotinylated immunopurified antibody against the recombinant protein.The secondary antibody used was HRP-streptavidin and ECL was used fordetection.

To quantitate the amount of OB in mouse serum, increasing amounts of therefolded recombinant mouse OB protein (0.01, 0.1, 0.5, 2.0, 15.0 ng)were added to 100λ of C57BL/6J ob/ob plasma and incubated at 4° C. for 3hours with the protein A sepharose conjugated antibody. After extensivewashing with buffer A (10 mM sodium phosphate buffer, pH 7.4; 100 mMNaCl; 1% Triton X-100, 5 mM EDTA, 1 mM PMSF), samples were resuspendedin sample buffer, loaded on a 15% SDS-PAGE and transferred to anitrocellulose membrane. Western blotting was performed using animmunopurified biotinylated anti-amino-terminus antibody as a primaryantibody and HRP-streptavidin as a secondary antibody, followed by ECLdetection. Cytoplasmic extracts were prepared by homogenizing adiposetissue in NDS buffer (10 mM Tris, pH 7.5, 10 mM NaCl, 60 mM ICCI, 0.15mM spermine, 0.5 mM spermidine, 14 mM P-mercaptoethanol, 0.5 m EGTA, 2mM EDTA, 0.5% NP-40) by polytron and dounce homogenization, and removalof nuclei was accomplished by centrifuging at 700 g.

Immunoprecipitations were performed as described above except thatimmunopurified anti-human OB antibodies were used. For the ELISA, 100 mlof a 1 mg/ml solution of immunopurified anti-human OB antibody wasdissolved in a borate buffered PBS solution and applied overnight tomicrotiter (Corning cat. #2595) plates at 4° C. The plates were thenwashed 4 times with borate saline solution containing 0.05% Tween 20 andexcess liquid was removed. Plates were blocked by incubation at roomtemperature for 2 hours with 240 ml per well of borate saline buffercontaining 0.3% gelatin and then washed and dried. Either known amountsof a refolded human OB protein or plasma samples in a 100 ml volume wereincubated in individual wells overnight at 4° C. After washing, theplates were incubated with 100 ml of a biotinylated immunopurifiedanti-human antibody (0.1 mg/ml in a gelatin-borate buffered solution)for 4 hours at room temperature. After washing, horseradish peroxidase(HRP)-streptavidin was added to the plates (0.1 mg/ml in borate buffer,0.3% gelatin). HRP substrate solution (ABTS, 0.3 mg/ml and H₂ O₂, 0.01%in citric acid) was then used for detection and the O.D. was measured at414 nM to quantitate the antibody binding.

The mouse and human OB gene coding sequences were PCR amplified fromplasmids containing OB cDNA sequences and subcloned into the pPIC.9plasmid (Invitrogen).

The human 5' primer used was

5' GTATCTCTCGAGAAAAGAGTGCCCATCCAAAAAGTCCAAG 3'(SEQ ID NO:34)

and the 3' primer was

5' GCGCGAATTCTCAGCACCCAGGGCTGAGGTC 3' (SEQ ID NO:35).

For mouse, the 5' primer was

5' GTATCTCTCGAGAAAAGAGTGCCTATCCAGAAAGTCCAGG 3'(SEQ ID NO:36)

and the 3' primer was

5' GCGCGAATTCTCAGCATTCAGGGCTAACATC 3' (SEQ ID NO:37).

The 5' primer for both mouse and human contains a XhoI site at the 5'end and coding sequences for the last 4 amino acids of the α-matingfactor signal sequence present in the vector pPIC.9. This vector directssecretion of heterologously expressed genes from the cell into theculture media. The 5' PCR primer also includes the first 19 nucleotidesof the OB gene open reading frame after the signal sequence cleavagesite, before the alanine at amino acid position 21. The 3' primercontains an EcoRI site at its 5' end, which is immediately followed bysequences complementary to the putative OB stop codon. The PCRconditions were as follows: denaturing for 1 min. at 94° C., annealingfor 1 min. at 55° C. and extension for 2.5 min. at 72° C. Low-cycle PCR(15 cycles) and the proof-reading polymerase PFU (Stratagene) were usedto limit the number of PCR-generated mutations. The PCR products weredigested with XhoI and EcoRI and cloned into similarly digested vector,pPIC.9. All constructs were sequenced on both strands to ensure theabsence of any PCR-generated mutations. Clones were transformed intoPichia pastonis (His⁻) by the spheroplast method and selected onhistidine deficient media. Approximately 200 mouse and human clones werescreened for high-copy number integration by a colony hybridizationassay. The high copy number clones were then assayed for OB expression,initially by Coomassie staining showing the presence of a novel 16 kDprotein present in the culture media of transformed yeast. The 16 kDband was confirmed to be OB using antibodies raised against thebacterially expressed OB protein. The recombinant proteins were purifiedby a two-step purification method described below. Mass spectrometry andcyanogen bromide treatment were performed as described in Beavis et al.,Proc. Natl. Acad. Sci. USA, 87:6873-6877 (1990).

The entire OB coding sequence of the mouse and human OB genes C-terminalto the signal sequence were subcloned into the pET15b expression vector(Novagen) and overexpressed in Escherichia coli [BL21(DE3)plYsS] usingthe T7 RNA polymerase system [Studier et al., Meth. Enzymology,185:80-89 (1990)]. Cells grown at 30° C. to an absorbency of 0.7 at 595nM and induced with 0.5 mM isopropyl-β-D-thiogalacto-pyranosideovernight were collected by low-speed centrifugation. Lysis wasperformed by three cycles of freeze thaw and DNA digestion was performwith DNaseI. Membrane extraction was performed by sonication anddetergent solubilization, and the final inclusion body pellet wasdissolved in 6M guanidine-HCl, 20 mM HEPES, pH8.4. Recombinant OBproteins were purified under denaturing conditions by IMAC using aNi-ion affinity column and washing with increasing amounts of imidazole.Purified denatured OB protein was then stored in 6 M guanidine-HCl, 10mM sodium acetate (NaAc), pH 5, and reduced using 1 mM DTT at roomtemperature for 1 hour. Denaturation was performed by diluting thereduced protein into 20% glycerol, 5 mM CaCl₂, 5 mM NaAc, pH 5, throughmixing and incubation at room temperature for 8-12 hours. Afterdenaturation the pH was adjusted to 8.4 by addition of Tris to 10 mM,and the hexa-histidine tag was removed by thrombin cleavage. Cleaved,renatured protein was repurified by IMAC to separate product fromthrombin and uncleaved fusion protein. Cleaved, renatured protein elutesfrom the Ni-ion affinity column at 40 mM imidazole, whereas thrombin isnot retained and uncleaved fusion protein elutes at 0.2 mM imidazole.Product was then concentrated, treated with 100 mM EDTA and 10 mMpotassium ferricyanide and further purified by gel filtration usingPharmacia superdex 75 16/60 column.

An Ellman's assay was conducted as described in Ellman, Arch. Biochem.Biophys., 82:70-77 (1959). Ellman's reagent was prepared by dissolving39.6 mg 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in 10 ml 0.05 Mphosphate, pH 8. A calibration curve was constructed in theconcentration range of 10-120 mM free sulfhydryl (using a 1 mM stocksolution of reduced DTT) at 412 nm. Each assay was performed using 0.02ml Ellman's reagent and a total reaction mixture of 0.5 ml. The measuredextinction coefficient was 12974 M⁻¹ cm⁻¹ for free sulfhydryl group(correlation coefficient 0.99987), which is within 5% of the previouslyreported value of 13600 M⁻ cm⁻¹.

Fifty ml of 2 mg/ml pure gel filtered protein, corresponding to apossible free sulfhydryl concentration of about 24 mM in the finalreaction mixture, was subjected to Ellman's assay. The resultingsolution gave A₄₁₂ of about 0.02, suggesting that the two cysteineresidues in the protein are in an oxidized state forming cystine or thattheir free sulfhydryl groups are completely buried within theinaccessible core of the folded protein. Identical results were obtainedby conducting the same assay on unfolded protein in the presence of 6 Mguanidine-HCl.

Mice were individually caged in a pathogen-free environment andacclimated to a diet containing 35% (w/w) Laboratory Rodent Diet 5001(PMP Feeds, Inc.), 5.9% (w/w) tapioca pudding mix (General Foods) and59.1% water, which has an energy content of 1.30 kcal/gm. The diet wassterilized by autoclave and packed into 60 mm plastic dishes, which werefixed to the tops of 100 mm petri dishes. Tapioca gives the diet a pastytexture making it difficult for the animal to spread the food in thecage. The 100 mm lid recovers the small amount of food spilled by theanimal. A fresh dish of food was placed into the cage each morning andthe previous day's dish was removed and weighed. The difference inweight provided a measure of daily food consumption. Effects ofrecombinant protein on food intake and body weight were measured inthree strains of mice: C57B1/6J ob/ob, C57 B1/Ks db/db and CBA/J +/+,purchased from the Jackson Laboratory. Thirty mice from each strain weredivided into groups of 10. One group from each strain received dailyintraperitoneal (i.p.) injections of the refolded bacterial ob proteinat a dose of 5 mg/g/day in 300 μL of PBS. A second group received i.p.injections of the same volume of PBS. These control mice receivedinjections of the PBS dialysate of the recombinant protein. The PBS wascleared of endotoxin using an Acticlean ETOX column. A third group ofanimals did not receive injections. Food intake was recorded daily andbody weight measurements were recorded regularly over a 3.5 weekinterval. For the pair feeding experiment, the food intake of a separategroup of ob mice was matched on a daily basis to that consumed by the obmice receiving protein.

Results

The OB Protein Circulates in Mouse, Rat and Human Plasma.

Recombinant mouse and human OB protein was prepared using the pET-15bbacterial 10 expression vector (Novagen) and by cloning into Pichiapastoris, a yeast expression system that secretes recombinant proteinsdirectly into the culture media. The ob protein expressed in yeastincludes the 146 amino acids carboxy-terminal to the signal sequence.Rabbits were immunized with the bacterial proteins (HRP, Inc.).Antibodies were immunopurified (Research Genetics) and used forimmunoprecipitations and Western blots of protein from plasma andadipose tissue.

The OB protein from mouse plasma migrates with an apparent molecularweight of 16 kD by SDS-PAGE. The electrophoretic mobility is identicalto the recombinant OB protein secreted by yeast after signal sequenceremoval (FIG. 24A) The protein was not detected in plasma from C57BL/6Job/ob mice that have a nonsense mutation at codon 105. Several differentantisera failed to identify the truncated 105 residue polypeptide chainpredicted by the cDNA sequence.

A ten-fold increase in the level of circulating protein was observed indb/db mice relative to a control animal (FIG. 24A). Immunoprecipitationof plasma from wild-type and fa/fa rats revealed a twenty-fold increasein the level of OB protein in the mutant rat compared to wild type (FIG.24B). The db mutation results in an obese phenotype identical to thatseen in ob mice (Bahary et al., 1990, supra). Fatty rats are obese as aresult of a recessive mutation in a gene homologous to db (Truett etal., 1991, supra). In order to quantitate the level of OB in mouseplasma, increasing amounts of recombinant protein were added to serumand immunoprecipitated (FIG. 24C). A linear increase of the signalintensity on Western blots was seen with increasing amounts ofrecombinant protein. Comparison of the signal intensity of the nativeprotein in mouse plasma to the standards indicated that the circulatinglevel of the OB protein in wild-type mice is approximately 20 ng/ml.These data demonstrate that the immunoprecipitations and Western blotswere performed under conditions of antibody excess. Increased levels ofthe OB protein were also seen in protein extracts of adipose tissue fromdb/db mice relative to controls (FIG. 24D). As expected for a secretedprotein, the protein from the adipose tissue fractionated with the crudemembrane fraction (data not shown).

Plasma samples from six lean human subjects with a Body Mass Index lessthan 25 (BMI=weight/length²) were immunoprecipitated usingimmunopurified antibodies to the human protein. The immunoprecipitatedmaterial migrated with an electrophoretic mobility identical to thatseen for the 146 amino acid human protein expressed in yeast. Theintensity of the signals varied significantly among the six samples(FIG. 25A). Densitometry of the autoradiograph revealed an approximatelyfive-fold difference in the levels in individuals HP1 and HP6, withintermediate levels in the other subjects. An enzyme linked immunoassay(ELISA) was developed using the immunopurified antibody and the refoldedbacterial protein as a standard (see below). The resulting standardcurve is shown in FIG. 25B. Using this assay, the plasma levels of theOB protein in the six human plasma samples varied between 2-15 ng/ml(FIG. 25C). The level of the OB protein in plasma from HP6 was outsideof the linear range of the immunoassay and is ≧15 ng/ml. Thesequantitative differences correlated with those seen on Western blots.

Preliminary data suggest that leptin may circulate, at least in part,complexed to another protein or proteins. This conclusion was based onheterogeneity of the shape of the titration curve for serum comparedwith recombinant standard. Analysis of a large amount of leptinimmunopurified on a rabbit anti-OB column by gel filtration HPLC underdenaturing and non-denaturing conditions, with monitoring by ELISA andSDS-PAGE suggested that the OB polypeptide behaved like a high molecularweight complex. However, these data remain preliminary; the OB bindingprotein, if any, has yet to be characterized.

Structural Features of the OB Protein.

Since the OB protein has two cysteine residues, it could form eitherintra- or intermolecular disulphide bonds under oxidizing conditions invivo. Western blots were repeated with and without the addition ofreducing agents to the sample buffer. Under both conditions, the OBprotein in human serum migrated as a monomer (data not shown). Undernonreducing conditions, protein immunoprecipitated from db mouse serumwas detected at positions consistent with that of both a monomer of 16kD and a dimer of approximately 32 kD (FIG. 26A). The higher molecularweight moiety disappeared under reducing conditions suggesting that afraction of mouse OB circulates as a higher molecular weight species viaformation of an intermolecular disulphide bond. Approximately 80% ofmouse OB circulates as the approximately 16 kD protein and 20% as theapproximately 32 kD form.

The same molecular forms are seen when the mouse and human proteins areexpressed in Pichia pastoris [Abrams et al., Immunol. Rev., :5-24(1992)]. In these studies, the DNA sequence corresponding to the 146amino acid mature OB protein was cloned downstream of the yeast a-matingfactor signal sequence in the pPIC.9 vector (Invitrogen). The OB proteinwas purified from the yeast media of strains expressing the mouse andhuman proteins and electrophoresed under reducing and nonreducingconditions (FIG. 26A). The mouse protein was expressed in yeast mainlyas a dimer under nonreducing conditions, and only as a monomer in thepresence of reducing agents. The recombinant human protein migrated tothe position of a monomer under both conditions (data not shown).

The purified human protein expressed in Pichia had a molecular mass of16,024±3 Da as determined by mass spectrometry 1990 (Beavis, 1990,supra). This value is in agreement with the mass calculated from theamino acid sequence of the protein containing a single intramoleculardisulfide bridge (16,024 Da). Matrix-assisted laser desorption massspectometric analysis of cyanogen bromide cleavage products of theprotein indicates that cysteines 117 and 167 are linked through anintramolecular disulphide bond (FIG. 26B) Cyanogen bromide cleavescarboxy-terminal to methionine residues.

Preparation and Characterization of Bioactive Recombinant Protein.

Mouse OB protein was expressed in E. coli from a pET-15b plasmid as aninsoluble fusion protein, with a 20 residue, N-terminal hexa-histidinetag containing a thrombin cleavage site. Bacterial inclusion bodies weresolubilized using guanidine-HCl and purified under denaturing conditionsusing immobilized metal ion affinity chromatography (IMAC) (FIG. 27).Purified, denatured fusion protein was reduced, diluted and permitted torefold in aqueous solution at room temperature. Following thrombincleavage, renatured mouse OB protein containing four additionalN-terminal residues (Gly-Ser-His-Met; SEQ ID NO: 38) was repurified byIMAC to >98% homogeneity, as judged by SDS-PAGE and mass spectrometry.Matrix-assisted laser desorption mass spectrometry gave a measured massof 16,414±3 Da (predicted mass =16,415 Da). Both reducing andnon-reducing SDS-PAGE gels demonstrated a single molecular species withapparent and molecular weight of 16 kD (data not shown).

Dynamic light scattering using a DP801 Molecular Size Detector (ProteinSolutions, Inc.) demonstrated that the renatured mouse OB protein waslargely monomeric, with some higher-order aggregates. The protein wastreated with EDTA and chemically oxidized. Higher molecular weightspecies were then removed by gel filtration. Further dynamic lightscattering confirmed that the purified, renatured recombinant mouse OBprotein was monodispersed. Following dialysis against phosphate bufferedsaline (PBS), bacterial endotoxin was removed using an Acticlean ETOXcolumn (Sterogene Bioseparations, Inc.). The final yield of protein was45 mg/L.

Ellman's assay was performed on the purified, renatured recombinantmouse OB protein to assess its oxidation state (Ellman, 1959, supra).Both renatured protein and protein unfolded by 6M guanidine-HCldemonstrated <0.5% free sulfhydryl content, demonstrating that themonomeric product contains an intramolecular disulphide bond. This wasconfirmed by mass spectrometry of the cyanogen bromide cleavage productsof the refolded bacterial protein (data not shown).

Bioactivity of the OB Protein.

The purified, renatured recombinant mouse OB protein was administered asa daily intraperitoneal injection of 5 mg/kg/day to groups of 10C57B1/6J ob/ob (age, 16 weeks), C57B1/Ks db/db (age, 12 weeks) and CBA/J+/+ (age, 8 weeks) mice. An equal number of animals received PBS as adaily injection. The PBS used for the control injections was derivedfrom the dialysate after equilibration of the protein. Ten additionalanimals from the three mouse strains did not receive injections. Thefood intake of individual animals was monitored daily and the weights ofthe animals were recorded at three or four day intervals. The cumulativeresults for food intake and body weight from each of the 9 groups ofmice are shown in FIG. 28A through F, and the statistical significanceof the data are shown in Table 1. The food intake of the C57B16J ob/obmice injected with protein was significantly decreased after the firstinjection and continued to decrease until the fifth day, when itstabilized at a level equal to approximately 40% of the intake of theanimals receiving injections of PBS (p<0.001). The sham injected ob micedid not lose weight over the three week study period. The C57B1/6J ob/obmice receiving protein lost approximately 10% of their body weight after5 days (p<0.001). These animals continued to lose weight over the threeweek treatment at which point the weight of the ob animals receivingprotein had decreased to an average of 60% of their initial body weight(p<0.0001). A separate group of ob mice were pair fed to the ob micereceiving protein. The data in FIG. 29B show that the pair fed mice lostsignificantly less weight than the animals receiving the recombinantprotein (p<0.02). A photograph of two mice receiving injections ofeither protein or vehicle shows the gross difference in appearanceresulting from the protein treatment (FIG. 29B). In order to furtherascertain the effects of the protein, autopsies of two mice in each ofthe groups were performed. Gross inspection of the ob mice receivingprotein revealed a dramatic decrease in body fat as well as the size ofthe liver. The liver weights of the db and wild-type mice were unchangedwith treatment. The livers from the ob mice receiving the injections ofPBS weighed 5.04 and 5.02 grams vs. 2.23 and 2.03 grams in the animalsreceiving the recombinant protein. In contrast to the pale fatty livercharacteristic of ob mice, the liver from the ob mice receiving proteinacquired the darker color characteristic of normal liver (FIG. 29C).Histologic sections of the liver indicated that the untreated animalshad a fatty liver that was markedly improved in protein treated animals(data not shown).

In contrast to the ob mice, there were no significant differences inbody weight or food intake in the C57BL/Ks db/db mice receiving proteinrelative to the control group receiving vehicle (FIG. 28A through F,Table 1). All three groups of db/db mice lost between 2-5 grams duringthe treatment period. The average blood glucose of the db mice wasmeasured using a glucometer, and was ≧500 mg/dl in all of the miceindicating that these animals had developed diabetes secondary toobesity. The injections of db mice were terminated after two weeks.

In wild-type mice there was a small but significant decrease in bodyweight following administration of the recombinant ob protein (FIG. 28Athrough F, Table 1). After five days of protein injection, the treatedmice lost an average of 0.5 grams while control mice gained 0.4 grams(p<0.02). At two subsequent time points, the animals receiving proteinweighed significantly less than the mice receiving daily injections ofPBS. The significance of the weight change was reduced at the later timepoints. In the animals that lost weight, the food intake was notsignificantly different from control animals. The injections of PBS hada small but significant effect on food intake and body weight in ob, dband wild-type mice as compared to mice not receiving injections(p<0.05).

                  TABLE 1                                                         ______________________________________                                        Animal                                                                              Treatment                                                                              WEIGHT CHANGE                                                  Group Group    Days   n   Mean     Std. Error                                                                            p                                  ______________________________________                                        ob/ob protein  1-5    10   -6.38000000                                                                           0.47628190                                                                            <0.001                                   vehicle          9   -0.14444444                                                                           0.24444444                                       protein  1-12   10  -14.45000000                                                                           0.70793126                                                                            <0.001                                   vehicle          9     0.98888889                                                                          0.38058597                                       protein  1-27    6  -24.28333333                                                                           0.69924563                                                                            <0.0001                                  vehicle          5     4.30000000                                                                          0.79874902                                 db/db protein  1-5    10   -1.47000000                                                                           0.36939891                                                                              0.240                                  vehicle         10   -2.00000000                                                                           0.23142073                                       protein  1-12   10   -3.75000000                                                                           0.77348418                                                                              0.610                                  vehicle         10   -4.19000000                                                                           0.34655447                                 CBA/J protein  1-5    10   -0.48000000                                                                           0.17876117                                                                              0.006                                  vehicle         10     0.38000000                                                                          0.21489015                                       protein  1-12   10   -0.12000000                                                                           0.45748103                                                                              0.015                                  vehicle         10     1.20000000                                                                          0.18378732                                       protein  1-27    5     1.98000000                                                                          0.48723711                                                                            <0.651                                   vehicle          6     2.23333333                                                                          0.20763215                                 ______________________________________                                    

Discussion

An endocrine function for the protein product of the OB locus was firstsuggested by Coleman, who showed that the body weight of ob/ob mice wasreduced after parabiotic union to normal or db mice (Coleman et al.,1978, supra). The results indicated above support this hypothesis byshowing that OB protein circulates in the bloodstream and thatinjections of recombinant protein reduce body weight. The molecularweight of the gene product encoded by the OB gene is approximately 16kD, which is equal to the 146 amino acid sequence carboxy-terminal tothe signal sequence. The recombinant OB protein is not modified whenexpressed in Pichia pastoris. Expression of mammalian genes in Pichiagenerally results in the formation of the correct protein structure[Cregg et al., Bio/Technology, 11:905-914 (1993)]. These findingssuggest that the OB protein is not glycosylated and is notpost-translationally processed in vivo. The data do not exclude thepossibility that the OB protein is noncovalently bound to itself orother proteins in plasma or adipose tissue. Although proteolyticcleavage of the protein has not been excluded, lower molecular weightforms of the OB protein were not detected by any of the antisera used,including four anti-peptide antibodies.

The OB protein has two cysteine residues and circulates as a monomer inhuman, and as a monomer and dimer in mouse. An intramolecular disulphidebond typical of secreted molecules is found when the human protein isexpressed in Pichia pastoris suggesting that it is likely to be presentin vivo. This is supported by the bioactivity of the recombinantbacterial protein, which has an intramolecular disulphide bond. Themouse OB protein can be found in plasma as a monomer and as a dimer. Themonomer and dimer are seen when the mouse OB protein is expressed inyeast showing that the propensity of the mouse protein to form a dimeris a result of differences in the primary sequence relative to the humanprotein. While it is clear that the monomer has bioactivity, thefunctional activity of the dimer is unknown.

The effect of the OB protein on food intake and body weight in ob miceis dramatic. After three weeks treatment, the ob mice receiving dailyinjections of recombinant protein had lost 40% of their weight and wereconsuming 40% as much food as control animals. Moreover, the weight ofthe treated ob mice had not yet equilibrated at the time the experimentwas terminated. The results of the pair feeding experiment indicateweight loss is a result of effects on both food intake and energyexpenditure. Thus, a separate group of ob mice whose caloric intake wasrestricted to that of ob mice receiving protein, lost significantly lessweight than the animals receiving protein. The reduction in food intakein ob/ob mice to a level lower than that of wild-type mice, within a dayof receiving the OB protein, indicates that they are especiallysensitive to its effects. Indeed, the OB receptor may be upregulated inthese animals. Food intake of treated ob mice became relatively constantafter five days of treatment. If this is the result of the proteinhaving reached steady state levels, it would suggest that the proteinhas a relatively long half-life [The Pharmacological Basis ofTherapeutics, pp. 19-45, Goodman and Gilman, eds., Pergamon Press, NewYork, (1990)]. This conclusion is consistent with data from parabiosisexperiments [Coleman et al., 1978, supra; Weigle, Int. J. Obesity,12:567-578 (1988)].

Effects of recombinant protein on the body weight of wild-type mice weresmall but statistically significant during the first two weeks of thestudy. While the difference in weight between wild-type mice receivingprotein vs. PBS was sustained at later time points, the statisticalsignificance of the data greatly diminished after three weeks. The earlyweight loss could not be accounted for by a difference in food intake.Presumably, the measurement of food intake was not precise enough todetect a decrease resulting in a one gram difference in body weightduring treatment. These observations differ from the results of previousexperiments in which wild-type rodents have been joined by parabioticunion to db mice, fa rats, rats with hypothalamic lesions and ratsrendered obese by a high calorie diet [Coleman et al., 1978, supra;Harris et al., 1987, supra; Harris et al., "Physiological and metabolicchanges in parabiotic partners of obese rats", in Hormones,Thermogenesis and Obesity, Lardy and Straatman, eds., Elsevier SciencePublishing Co., New York (1989); Hervey, J. Physiol., 145:33& 352(1959)]. In each case, the wild-type animals become anorectic and losecopious amounts of weight. As the levels of OB protein are increased indb mice and fa rats and the level of OB RNA is increased in mice withhypothalamic lesions, it is likely that wild-type mice can respond to OBwhen it circulates in plasma at a sufficiently high level. The findingsreported here are consistent with the possibility that the levels of theadministered protein were below endogenous levels, leading toequilibration at a slightly lower body weight. Quantitation of thecirculating levels of the OB protein in the treated mice will resolvethis issue. While an immunoassay of the mouse protein is not yetavailable, immunoprecipitations have suggested that the levels of thecirculating OB protein were not substantially elevated in the wild-typemice receiving protein.

The lesser effect of the protein on wild-type mice and the absence of aresponse in db mice makes it unlikely that the treatment has nonspecificor aversive effects. All of the db mice lost a small amount of weightduring the treatment period, whether or not they were receiving the obprotein. The db animals were markedly hyperglycemic and the weight lossis likely to be the result of diabetes and not the experimentalprotocol. C57BL/Ks db/db mice often develop diabetes and begin to losesmall amounts of weight when of the age of the animals used in thisstudy (Coleman et al., 1978, supra). C57B1/6J ob/ob mice of a similarage do not develop significant hyperglycemia. These phenotypicdifferences are thought to be the result of genetic differences in thestrains (C57B16J vs. C57B1/Ks) carrying the mutations (Coleman et al.,1978, supra).

The failure to detect the truncated 105 amino acid protein predicted bythe cDNA sequence of the OB gene in C57B1/6J ob/ob mice suggests thatthe mutant protein is either degraded or not translated. However, thepossibility that the antisera used do not detect this truncated proteincannot be excluded. The observed ten-fold increase in the levels of theOB protein in db mice compared to wild type suggests that the OB proteinis overproduced when there is resistance to its effects. These datacorrelate with studies of the OB mRNA. As mentioned, previousexperiments have shown that mutations of the mouse db and the rat fagenes, which map to homologous chromosomal regions, result inoverproduction of a plasma factor that suppresses body weight (Truett etal., 1991, supra; Coleman, 1978, supra; Hervey, 1959, supra). In bothcases, it has been suggested that the mutant animals are resistant tothe effects of the OB protein. This possibility is confirmed by theobservation that the OB protein has no effect on body weight or foodintake when administered to db mice.

Obesity in humans could be associated with increased levels of the OBprotein in plasma in individuals who are relatively unresponsive to thehormone. On the other hand, reduced expression of OB could also lead toobesity in which case "normal" (i.e., inappropriately low) levels of theprotein might be found. Thus, the levels of OB protein in human plasmacould be a marker for different forms of obesity. In a small group oflean subjects with BMI <25, low nanogram levels of circulating OBprotein are detectable by ELISA. Significantly, variable concentrationswere noted suggesting that the level of expression and/or sensitivity tothe protein may play a role in determining body weight.

The site of action of the OB protein is unknown. The protein affectsboth food intake and energy expenditure, a finding consistent withclinical studies indicating that alterations of both systems act toregulate body weight [Leibel et al., N. Engl. J. Med., 332:621-628(1995); Keesey et al., in Association for Research in Nervous and MentalDisease, pp. 87-96, Stunkard and Stellar, eds., Raven Press, New York.(1984)]. The hypothalamus is likely to be downstream of OB in thepathway that controls body weight, although direct effects on a varietyof organs are possible.

EXAMPLE 9: Increased Expression in Adipocytes of OB RNA in Mice withLesions of the Hypothalamus and with Mutations at the db Locus

The gene product of the recently cloned mouse obese gene (OB) plays animportant role in regulating the adipose tissue mass. OB RNA isexpressed specifically by mouse adipocytes in vivo in each of severaldifferent fat cell depots including brown fat. It is also expressed incultured 3T3-442A preadipocyte cells that have been induced todifferentiate. Mice with lesions of the hypothalamus, as well as micemutant at the db locus, express a twenty-fold higher level of OB RNA inadipose tissue. These data suggest that both the db gene and thehypothalamus are downstream of the OB gene in the pathway that regulatesthe adipose tissue mass and are consistent with previous experimentssuggesting that the db locus encodes the OB receptor. In the db/db andlesioned mice, quantitative differences in the level of expression of OBRNA correlated with the lipid content of adipocytes. The molecules thatregulate the level of expression of the OB gene in adipocytes are likelyto play an important role in determining body weight, as are themolecules that mediate the effects of OB at its site of action.

Materials and Methods

In Situ Hybridization.

White fat tissues from identical abdominal regions of wild type (wt) anddb mice were processed simultaneously according to the modified methoddescribed by Richardson et al., Growth, Development & Aging, 56:149-157(1992). Briefly, tissues were fixed in Bouin's solution for 2 hours at4° C. They were then dehydrated by serial treatment of increasingconcentrations of ethanol from 10% to 100%, each for 5 min. at 4° C.Further incubation of tissues with xylene (1 hr.) and paraffin (2 hr.)were performed at 65° C. Embedded wt and db/db fat tissues weresectioned and mounted by the same conditions later. Sections were bakedat 65° C. for 1 hr. and treated with xylene and serial dilutions ofethanol from 100% to 50%, each for three min. at room temperature. Anantisense RNA probe of OB gene was synthesized by in vitro transcriptionof linearized OB gene coding sequence upstream of a Sp6 RNA polymerasepromoter. In situ hybridization was carried out exactly according toSchaeren-Wiemers et al. Histochemistry, 100:431-440 (1993).

RNA Preparation and Cell Culture.

Total RNA and Northern blots were prepared as described. Stromalvascular cells and adipocytes were prepared according to Rodbell, andRNA from both fractions was prepared according to Dani et al., Mol.Cell. Endocrinol., 63:199-208 (1989); Rodbell, J. Biol. Chem.239:375-380 (19). After sub-cloning, 3T3-F442 cells were grown inDulbecco's modified Eagle medium containing 10% foetal bovine serum(defined as standard medium) [Dani et al., "Molecular biology techniquesin the study of adipocyte differentiation", in Obesity in Europe vol 88,pp. 371-376, Bjorntorp and Rossner, Eds., John Libbey Company Ltd.,London, England (1989)]. At confluence, cells were treated in standardmedium supplemented with 2 nM triiodothyronine (T3) and 17 nM insulin.Twelve days later, RNA was prepared as above.

Gold ThioGlucose Treatment.

One month old female CBA/J mice were treated with a singleintraperitoneal injection of aurothioglucose (Sigma catalog no. A0632)at a dose of 2.0 mg/g in normal saline. Control animals were injectedwith normal saline. Mice were weighed one month after the treatment.Adipose tissue RNA was isolated from those treated animals whose weighthad increased more than twenty grams post-GTG treatment.

Results

The OB gene was recently found to be expressed in adipose tissue [Zhanget al., Nature, 372:425-432 (1994)]. As adipose tissue is composed ofmany cell types including adipocytes, preadipocytes, fibroblasts andvascular cells, in situ hybridization was performed to sections ofepididymal fat pads from normal animals with sense and antisense OBriboprobes [Richardson et al., 1992, supra; Wasserman, "The concept ofthe fat organ: in Rodahl, Issekutz, fat as a tissue", pp. 22-92, McGrawHill, New York (1964)]. When using the antisense probe, positive signalswere detectable in all of the adipocytes in the section (FIG.30--labeled Wt). Signals were not noted when the antisense probe washybridized to sections of brain (data not shown). Hybridization of theantisense probe to sections of adipose tissue from C57B1/Ks db/db micewas greatly increased, confirming the adipocyte specific expression ofOB RNA and demonstrating a large increase in the level of OB RNA peradipocyte in these animals (FIG. 30--labeled db/db). Mice mutant at thedb locus are massively obese as part of a syndrome that isphenotypically identical to that seen in C57B1/6J ob/ob mice (Bahary etal., 1990, supra).

OB RNA was not synthesized by adipose tissue stromal cells separatedfrom adipocytes. As expected, cells in the adipocyte fraction expressedOB RNA using Northern blots (FIG. 31). The same result was obtainedusing RT-PCR (data not shown). These data support the conclusion thatonly adipocytes express the OB gene. Data from cultured adipocytesconfirm this conclusion. In these studies, 3T3-F442A cells were culturedusing conditions that lead to lipid accumulation, as part of a cellularprogram leading to differentiation into adipocytes. OB RNA was notexpressed in exponentially growing cells, nor in confluent 3T3-F442Apreadipocyte cells, which express early markers, while differentiationof these cells into adipocytes led to the expression of detectablelevels of OB RNA (FIG. 31) [Dani et al., J. Biol. Chem., 264:10119-10125(1989)]. The level of OB RNA is extremely sensitive to the cultureconditions, as no message was observed in late, post-confluent cells notexposed to insulin.

Hybridization studies showed that OB RNA is expressed in vivo in severaldifferent fat depots including the epididymal, parametrial, abdominal,perirenal, and inguinal fat pads (FIG. 32A). The precise level ofexpression in each of the depots was somewhat variable, with inguinaland parametrial fat expressing lower levels of OB RNA. OB RNA is alsoexpressed in brown adipose tissue, although the level of expression isapproximately 50-fold lower in brown fat relative to the other adiposetissue depots. These quantitative differences correlate loosely withpreviously reported differences in cell size among the different fatcell depots [Johnson et al., J. Lipid Res., 13:2-11 (1972)]. The amountof OB RNA in brown fat is unaffected by cold exposure (FIG. 32B). Inthis experiment, the level of uncoupling protein RNA (UCP) increased inbrown fat after cold exposure while the level of OB RNA did not change[Jacobsson et al., J. Biol. Chem., 260:16250-16254 (1985)]. Inaggregate, these data confirm that all adipocytes are capable ofproducing OB RNA and demonstrate a variable level of expression indifferent fat depots. These data support the possibility that the levelof the encoded protein correlates with the total adipose tissue mass.

Levels of OB RNA in db/db mice and mice with lesions of the hypothalamuswere measured. Lesions of the ventromedial hypothalamus (VMH) result inobesity as part of a syndrome resembling that seen in ob/ob and db/dbmice [Bray et al., Metabolism, 24:99-117 (1975)]. Parabiosis experimentssuggest such lesions result in over expression of a blood-borne factorthat suppresses food intake and body weight (Hervey, 1959, supra).Similar results are noted when mice mutant at the db locus areparabiosed to normal mice, suggesting that the OB receptor may beencoded by the db locus (Coleman et al., 1978, supra). Thus, obesityresulting from VMH lesions and the db mutation may be the result ofresistance to the effects of the OB protein. If so, a secondary increasein the levels of OB RNA in adipose tissue would be predicted.

Hypothalamic lesions were induced in female CBA mice using the chemicalgold thioglucose (GTG) [Debons et al., Fed. Proc., 36:143-147 (1977)].This treatment results in specific hypothalamic lesions, principally inthe ventromedial hypothalamus (VMH), with the subsequent development ofobesity within several weeks. Usually, a single intraperitonealinjection of GTG of 2.0 mg/gm body weight results in the development ofobesity within four weeks. One month old female CBA/J mice (20-25 grams)were treated with GTG and the subsequent weight gain of treated andcontrol animals is shown (Table 2). Adipose tissue RNA was prepared fromdb/db mice and from those GTG treated animals that gained >20 gm.Northern blots showed a twenty-fold increase in the level of OB RNA intwo month old db/db and GTG-treated mice compared to normal animals(FIG. 33).

                  TABLE 2                                                         ______________________________________                                        Weight Gain in Gold Thioglucose Treated Mice                                             control (n = 41)                                                                           GTG (n = 93)                                          ______________________________________                                        <10 g        41, (100%)      4, (4%)                                          10 g-20 g     0, (0%)       15, (16%)                                         >20 g         0, (0%)       74, (80%)                                         ______________________________________                                    

One month old female CBA/J mice were treated with gold thioglucose(GTG). Gold thioglucose (Sigma A0632) was administered intraperitonealyin normal saline solution at a dosage of 2.0 mg/g. Body weight ofcontrol and injected animals was recorded before and one month after theinjection. Animals were housed five to a cage and were fed ad libitum.The amount of weight gained one month post-injection is shown in Table2. Animals with a body weight gain greater that 20 g one month afterinjection were selected for further study.

Discussion

The gene product of the mouse OB gene circulates in mouse and humanplasma where it may act to regulate the adipose tissue mass. Furtherstudies on the regulation of expression and mechanism of action of OBwill have important implications for our understanding of thephysiologic pathway that regulates body weight.

The present Example shows that the OB gene product is expressedexclusively by adipocytes in all adipose tissue depots. This result isconsistent with the possibility that the protein product of the OB genecorrelates with the body's lipid stores. Moreover OB RNA is upregulatedtwenty-fold in db mice and mice with hypothalamic lesions. In theseanimals, the actual increase in the level of OB RNA per cell is likelyto be even higher than twenty-fold since the adipocyte cell size isincreased approximately five-fold in these animals (see FIG. 30) (Debonset al., 1977, supra). These data position the db gene and thehypothalamus downstream of OB in the pathway that controls body weightand is consistent with the hypothesis that the OB receptor is encoded atthe db locus (Coleman et al., 1978, supra). The molecular cloning of theOB receptor and/or the db gene will resolve this issue. The increase inthe level of OB RNA in db/db and GTG-treated mice also suggests a noncell-autonomous function of the OB gene product in fat cells [Ashwell etal., Proc. R. Soc. Lond., 195:343-353 (1977); Ashwell et al.,Diabetologia, 15:465470]. Thus, if the encoded protein acted directly onfat cells to inhibit growth or differentiation, the overexpression ofthe wild-type OB gene in GTG treated mice would result in a leanphenotype.

The most parsimonious explanation of these data is that the OB proteinfunctions as an endocrine signaling molecule that is secreted byadipocytes and acts, directly or indirectly, on the hypothalamus. Directeffects on the hypothalamus would require that mechanisms exist to allowpassage of the OB gene product across the blood brain barrier.Mechanisms involving the circumventricular organ and/or specifictransporters could permit brain access of a molecule the size of thatencoded by the OB gene [Johnson et al., FASEB J., 7:678-686 (1983);Baura et al., J. Clin. Invest., 92:1824-1830 (1993); Pardridge,Endocrine Reviews, 7:314-330 (1986)]. However, this hypothesis must beconsidered with caution until the means by which the protein might crossthe blood brain barrier have been identified. Moreover, possible effectson other target organs will need to be evaluated.

The fat cell signal(s) that are responsible for the quantitativevariation in the expression level of the OB gene is not yet known butcorrelates with differences in adipocyte cell size. Adipocytes fromdb/db mice are five times as large as those from normal mice, with acell size of approximately 1.0 μg lipid/cell (Johnson et al., 1972,supra). Prior evidence has indicated that fat cell lipid content and/orsize is an important parameter in determining body weight [Faust et al.,Am. J. Physiol., 235:279-286 (1978); Faust et al., Science, 197:393-396(1977)]. It could be that each fat cell expresses a low level of OB RNAthat further increases in proportion to the cell size. It is alsopossible that cell size is not the sensed parameter, but merelycorrelates with the intracellular signal that increases the expressionof the OB gene in adipocytes from db/db and VMH-lesioned mice. In anycase, the components of the signal transduction pathway regulating thesynthesis of OB RNA are likely to be important in determining bodyweight. Genetic and environmental influences that reduce the level ofexpression of OB would act to increase body weight, as would influencesthat decreased sensitivity to the encoded protein. The specificmolecules that regulate the level of expression of the OB gene are asyet unknown, and await a determination of the level(s) of gene controlthat leads to quantitative variation in the level of OB RNA, and anexamination of the regulatory elements of the OB gene. Theidentification of the molecules that regulate the expression of the OBgene in adipocytes, and those that mediate the effects of the encodedprotein at its site(s) of action, will greatly enhance our understandingof the physiologic mechanisms that regulate body weight.

EXAMPLE 10: RNA Expression Pattern and Mapping on the Physical,Cytogenetic, and Genetic Maps of Chromosome 7

OB RNA is expressed at high levels in human adipose tissue, and atsubstantially lower levels in placenta and heart. The human OB gene mapsto a large yeast artificial chromosome (YAC) contig derived fromchromosome 7q31.3. In addition to confirming the relative location ofthe gene based on mouse-human comparative mapping, this study hasidentified 8 established microsatellite markers in close physicalproximity to the human OB gene. Since mutations in mouse OB can resultin a syndrome that closely resembles morbid obesity in humans, thesegenetic markers represent important tools for studying the possible roleof the OB gene in inherited forms of human obesity.

Materials and Methods

Northern Blot Analysis.

Total RNA was prepared from adipose tissue using the method of Chirgwinet al., Biochem., 18:5294-5299 (1979). Northern blots, radiolabeling,and hybridizations were performed as described (Zhang et al., 1994,supra). Northern blots of polyA⁺ RNA (human MTN, human MTN II, and humanfetal MTN II) were obtained from CLONETECH (Palo Alto, Calif.), as werePCR primers used to generate the radiolabeled human actin probe.

STS Development.

Sequence tagged-site (STS)-specific PCR assays were developed andoptimized essentially as described [(Green et al., PCR Methods Applic.,1991; Green et al., Genomics, 11:548-564 (1991); Green, "Physicalmapping of human chromosomes: generation of chromosome-specificsequence-tagged sites", in Methods in Molecular Genetics Vol. 1, Geneand Chromosome Analysis (Part A), pp. 192-210, Adolph ed., AcademicPress, Inc., San Diego (1993); Green et al., Hum. Mol. Genet., 3:489-501(1994)]. Each STS is named using the prefix `sWSS` followed by a uniquenumber. Details about the 19 STSs reported here are provided in Table 3,with additional information (e.g., PCR reaction conditions, complete DNAsequence) available in GenBank and/or the Genome Data Base (GDB). Forthe microsatellite-specific STSs, the oligonucleotide primers used inthe PCR assays (Table 3) corresponded either to those employed forgenotype analysis (Table 4), or those designed (most often with thecomputer program OSP) [Hillier et al., PCR Methods Applic., 1:124-128(1991)] using the DNA sequence available in GenBank.

Table 3 illustrates STSs in the YAC contig containing the human OB gene

The 19 chromosome 7-specific STSs mapped to the YAC contig containingthe human OB gene (FIG. 35) are listed. In each case, the designated`sWSS` name, relevant alias, GDB-assigned locus name, STS source, PCRprimer sequences, STS size, and GDB identification number are indicated.The sources of STSs are as follows: `YAC End` (isolated insert end of aYAC) (Green, 1993, supra), `Lambda Clone` (random chromosome 7-specificlambda clone) (Green et al. 1991, supra; Green, 1993, supra), `GeneticMarker` (microsatellite marker, see Table 2) (Green et al. 1994, supra),`YAC Insert` (random segment from YAC insert), and `Gene` (gene-specificSTS). Note that for some genetic marker-specific STSs, the PCR primersused for identifying YACs (listed in this table) are different fromthose used for performing genotype analysis (Table 4), since thedetection of YACs containing a genetic marker does not requireamplification of the polymorphic tract itself. All of the indicated PCRassays utilized an annealing temperature of 55° C., except for sWSS494,sWSS883, sWSS1529, and sWSS2619 (which used 50° C.), sWSS999 and sWSS1174 (which used 60° C.), and sWSS808 (which used 65° C.). Additionaldetails regarding the STS-specific PCR assays are available in GDB.

                                      TABLE 3                                     __________________________________________________________________________                                             Size                                                                             SEQ ID                            STS Name                                                                            Alias  Locus                                                                                Source                                                                                 PCR Primers*                                                                                    NO:bp)                                                                           GDB ID                      __________________________________________________________________________                                                    No.                           sWSS1734    D7S2185                                                                            YAC End  CAAGACAAATGAGATAAGG                                                                           72                                                                              39  G00455-235                                              AGAGTTACAGCTTTACAG                                                                              40                                sWSS494     D7S2016                                                                             Lambda Clone                                                                           CTAAACACCTTTCCATTCC                                                                            112                                                                            41    G00-334-404                                          TTATATTCACTTTTCCCCTCTC                                                                          42                                sWSS883                                                                            UT528  D7S1498                                                                            Genetic Marker                                                                         TGCAGTAAGCTGTGATTGAG                                                                           490                                                                             43   G00-455-262                                           GTGCAGCTTTAATTGTGAGC                                                                            44                                sWSS2359                                                                           AFMa065zg9                                                                           D7S1873                                                                            Genetic Marker                                                                         AGTGTTGTGTTTCTCCTG                                                                             142                                                                            45     G00-455-247                                          AAAGGGGATGTGATAAGTG                                                                             46                                sWSS2336                                                                           AFMa125wh1                                                                            D7S1874                                                                            Genetic Marker                                                                        GGTGTTACGTTTAGTTAC                                                                            112                                                                             47   G00-455-244                                            GGAATAATGAGAGAAGATTG                                                                            48                                sWSS1218                                                                           AFM309yf1                                                                             D7S680                                                                             Genetic Marker                                                                        GCTCAACTGACAGAAAAC                                                                            154                                                                             49     G00-307-733                                          GACTATGTAAAAGAAATGCC                                                                            50                                sWSS1402                                                                                  D7S1916                                                                                 YAC End                                                                                  AAAGGGCTTCTAATCTAC                                                                       5137                                                                               G00-344-044                                            CCTTCCAACTTCTTTGAC                                                                              52                                sWSS999                                                                                   D7S1674                                                                                  YAC Insert                                                                           TAAACCCCCTTTCTGTTC                                                                           535                                                                                 G00-334-839                                          TTGCATAATAGTCACACCC                                                                             54                                sWSS1751    D7S2186                                                                                YAC End                                                                                   CCAAAATCAGAATTGTCAGAAG                                                                186                                                                              55  G00-455-238                                             AAACCGAAGTTCAGATACAG                                                                            56                                sWSS1174                                                                           AFM218xf10                                                                            D7SS514                                                                               Genetic Marker                                                                     AATATCTGACATTGGCAC                                                                               574                                                                              G00-307-700                                             TTAGACCTGAGAAAAGAG                                                                              58                                sWSS2061                                                                                  D7S2184                                                                                YAC End                                                                                   GTTGCACAATACAAAATCC                                                                      200                                                                           59  G00-455-241                                             CTTCCATTAGTGTCTTATAG                                                                            60                                sWSS2588                                                                                  D7S2187                                                                              YAC End                                                                                      ATCACTACACACCTAATC                                                                      117                                                                           61  G00-455-253                                             CCATTCTACATTTCCACC                                                                              62                                sWSS808                                                                             Pax-4       Gene      GGCTGTGTGAGCAAGATCCTAGGA                                                                     153                                                                            63    G00-455-259                                           TTGCCAGGCAAAGAGGGCTGGAC                                                                         64                                sWSS1392                                                                            AFM206xc1                                                                               D7S635                                                                            Genetic Marker                                                                      CTCAGGTATGTCTTTATC                                                                           75 65    G00-307-815                                           TGTCTCTGCATTCTTTTC                                                                              66                                sWSS1148                                                                             AFM199xh12                                                                          D7S504                                                                                 Genetic Marker                                                                    GACACATACAAACACAAG                                                                            60                                                                              67    G00-307-652                                           ATTGAGTTGAGTGTAGTAG                                                                             68                                sWSS1529                                                                                  D7S1943                                                                                 YAC End                                                                                  CAGGGATTTCTAATTGTC                                                                      116                                                                            69    G00-334-119                                           AAAAGATGGAGGCTTTTG                                                                              70                                sWSS2619                                                                           OB       OB Gene               CGTTAAGGGAAGGAACTCTGG                                                                 106                                                                            71 G00-455-256                                             TGGCTTAGAGGAGTCAGGGA                                                                            72                                sWSS404                                                                                   D7S1956                                                                                 Lambda Clone                                                                       ACCAGGGTCAATACAAAG                                                                           122                                                                             73    G00-334-251                                           TAATGTGTCCTTCTTGCC                                                                              74                                sWSS2367                                                                           AFMa345wc9                                                                            D7S1875                                                                               Genetic Marker                                                                     CAATCCTGGCTTCATTTG                                                                            81                                                                              75    G00-455-250                                           AAGGTGGGTAGGATGCTA                                                                              76                                __________________________________________________________________________     *The PCR Primers correspond consecutively, in seriatum, to SEQ ID NO: 39      through SEQ ID NO: 76.                                                   

    __________________________________________________________________________                       SEQ ID                                                     Marker Name                                                                           Type                                                                              Locus                                                                                    NO:ers*          GDB ID No.                            __________________________________________________________________________    UT528  Tetra.                                                                            D7S1498                                                                            TGCAGTAAGCTGTGATTGAG                                                                       77  G00-312-446                                                  GTGCAGCTTTAATTGTGAGC                                                                                 78                                     AFMa065zg9                                                                            (CA).sub.n                                                                        D7S1873                                                                               AGCTTCAAGACTTTNAGCCT                                                                    79 G00-437-253                                                  GGTCAGCAGCACTGTGATT                                                                                   80                                    AFMa125wh1                                                                            (CA).sub.n                                                                        D7S1874                                                                               TCACCTTGAGATTCCATCC                                                                        81                                                                            G00-437-263                                                  AACACCGTGGTCTTATCAAA                                                                                 82                                     AFM309yf10                                                                            (CA).sub.n                                                                        D7S680                                                                                 CATCCAAGTTGGCAGTTTTT                                                                     83                                                                             G00-200-283                                                  AGATGCTGAATTCCCAGACA                                                                                 84                                     AFM218xf10                                                                            (CA).sub.n                                                                        D7S514                                                                                 TGGGCAACACAGCAAA                                                                          G00-488-404                                                  TGCAGTTAGTGCCAATGTCA                                                                                 86                                     AFM206xc1                                                                              (CA).sub.n                                                                       D7S635                                                                                 CCAGGCCATGTGGAAC                                                                           G00-199-240                                                 AGTTCTTGGCTTGCGTCAGT                                                                                 88                                     AFM199xh12                                                                            (CA).sub.n                                                                        D7S504                                                                                 TCTGATTGCTGGCTGC                                                                           G00-488-280                                                 GCGCGTGTGTATGTGAG                                                                                 90                                        AFMa345wc9                                                                            (CA).sub.n                                                                        D7S1875                                                                               AGCTCTTGGCAAACTCACAT                                                                      91                                                                             G00-437-259                                                  GCCTAAGGGAATGAGACACA                                                                               92                                       __________________________________________________________________________     *The Primers correspond consecutively, in seriatum, to SEQ ID NO: 77          through SEQ ID NO: 92.                                                   

The human OB-specific STS (sWSS2619) was designed using DNA sequenceobtained from the 3' untranslated region of the cDNA. The humanPax4-specific STS (sWSS808) was developed using the following strategy.Oligonucleotide primers specific for the mouse Pax4 gene(GGCTGTGTGAGCAAGATCCTAGGA) (SEQ ID NO: 63) and(GGGAGCCTTGTCCTGGGTACAAAG) (SEQ ID NO: 93) [Walther et al., Genomics11:424-434 (1991)] were used to amplify a 204-bp fragment from humangenomic DNA (which was the same size product as that generated frommouse genomic DNA). This PCR assay was not suitable for identifyingcorresponding YACs, since a similarly-sized (200-bp) product was alsoamplified from yeast DNA. However, DNA sequence analysis of the PCRproduct generated from human DNA revealed substitutions at 20 positionsamong the 156 bases analyzed (data not shown). Using this human-specificsequence, a new primer (TTGCCAGGCAAAGAGGGCTGGAQ (SEQ ID NO: 64) wasdesigned and used with the first of the above mouse Pax4-specificprimers (see Table 3). The resulting human Pax4-specific PCR assay didnot amplify a significant product from yeast DNA and was thus used foridentifying corresponding YACs.

Identification of YACs by PCR-based screening.

Most of the YACs depicted in FIG. 35 were derived from a collection ofclones highly enriched for human chromosome 7 DNA (the `chromosome 7 YACresource`) (Green et al., 1995, supra) using a PCR-based screeningstrategy [Green et al., 1995, supra; Greena et al., Proc. Natl. Acad.Sci. USA, 87:1213-1217 (1990)]. In a few cases, clones were isolated byPCR-based screening (Greena et al., 1990, supra) of available totalhuman genomic YAC libraries constructed at CEPH [Dausset et al., BehringInst. Mitt., 91:13-20 (1992); Albertsen et al., Proc. Natl. Acad. Sci.USA, 7:4256-4260 (1990)] or ICI [Anand et al., Nucl. Acids Res.,17:3425-3433 (1989); Anand et al., Nucl. Acids Res. 18: 1951-1956(1990)]. Each YAC is named using the prefix `yWSS` followed by a uniquenumber.

Results and Discussion

Examination of the tissue expression of the human OB gene by Northernblot analysis revealed that OB RNA is expressed at a high level in humanadipose tissue and much lower levels in placenta and heart (FIG. 34).The size of the RNA (approximately 4.5 kb) was equivalent in human andmouse as well as in each of the expressing tissues. In these studies,five-fold higher signals were seen in 10 μg of total adipose tissue RNA,as in 2 μg of poly A⁺ placental RNA. A five-fold lower signal was seenin poly A⁺ RNA from heart compared to placenta. It is estimated that thelevel of OB RNA is approximately 250-fold lower in placenta than inadipose tissue. In this experiment, OB RNA was not detected in any ofthe other tissues analyzed, including brain, lung, liver, skeletalmuscle, kidney, and pancreas. Additional experiments did not reveal OBRNA in spleen, thymus, prostate, testis, ovary, small intestine, colon,peripheral blood leukocytes, or in fetal brain, liver, or kidneys (datanot shown). It is possible that OB is expressed at an undetectable level(by Northern blot analysis) in these latter tissues or in other tissuesthat were not studied. The observed pattern of expression in humandiffers somewhat from mouse, in which OB RNA is detected almostexclusively in adipose tissue.

Comparative mapping of the OB gene region in the mouse and humangenomes. The mouse OB gene is located on proximal chromosome 6 in aregion homologous with a portion of human chromosome 7q. Genes withinthis segment include (from proximal to distal): the Met protooncogene,the cystic fibrosis transmembrane conductance regulator (Cftr), pairedbox-containing gene 4 (Pax4), OB, and carboxypeptidase A (Cpa) (Zhang etal., 1994, supra; Friedman et al., 1991, supra). In the mouse, geneticmapping was used to demonstrate that Pax4 is tightly linked to OB(Walther et al., 1991, supra; Zhang et al., 1994, supra). The physicaldistance between OB and Pax4 was found to be approximately one megabasepairs (Mb) (Zhang et al. 1994, supra). Based on these comparativemapping studies, it was expected that the human OB gene would residebetween Pax4 and CPA on chromosome 7q. Furthermore, since human CFIR[Heng et al., Cell Genet., 62:108-109 (1993)] and Pax4 [Tamura et al.,Cytogenet. Cell Genet., 66:132-134 (1994)] were mapped by fluorescencein situ hybridization (FISH) to 7q31.3 and 7q32, respectively, the mostlikely cytogenetic position of the human OB gene would be in thevicinity of the 7q31.3-q32 boundary.

Mapping the OB gene on human chromosome 7.

An STS (sWSS2619) amplifying a small segment of the 3' untranslatedregion of the human OB gene was used to screen a collection of YACclones that is highly enriched for human chromosome 7 DNA (Green et al.,1995, supra) and 9 YACs were identified (yWSS691, yWSS1332, yWSS1998,yWSS2087, yWSS3319, yWSS3512, yWSS4875, yWSS4970, and yWSS5004). Toverify that these YACs contain the authentic human OB gene, twoadditional experiments were performed. First, each of the YACs wastested with a second human OB-specific PCR assay, and all were found tobe positive (data not shown). Second, yeast DNA from each clone wasdigested with EcoRI and analyzed by gel-transfer hybridization, using ahuman OB cDNA-derived probe. In all instances, a single hybridizing bandwas seen; and this band was the same size in the YACs and a P1 cloneknown to contain the human OB gene (data not shown).

Using the computer program SEGMAP (Green and Green, 1991, supra) andother YAC-based STS-content data that we have generated for chromosome 7(Green et al. 1991, supra; Green et al. 1994, supra; Green et al. 1995,supra), the human OB gene was found to reside within the YAC contigdepicted in FIG. 35. Specifically, this contig consists of 43overlapping YACs and 19 uniquely-ordered STSs. Details about each of the19 STSs are provided in Table 3. In addition to the OB-specific STS, thecontig also contains an STS (sWSS808) specific for the human Pax4[Tamura et al. 1994, supra; Stapleton et al, Nature Genet., 3:292-298(1993)], 7 STSs derived from chromosome 7-specific YACs, 2 STSs derivedfrom chromosome 7-specific lambda clones, and, importantly, 8microsatellite-specific STSs. Additional details about these 8 geneticmarkers, including sequences of the primers used for genotype analysis,are provided in Table 2. Of note, there is redundant YAC-basedconnectivity throughout the contig (i.e., there are 2 or more YACsconnecting each adjacent pair of STSs), lending strong support for therelative order of STSs shown in FIG. 35.

As depicted in FIG. 35, the predicted orientation of the humanOB-containing YAC contig is such that sWSS1734 is the centromeric-mostSTS (i.e., closest to CFTR) whereas sWSS2367 is the telomeric-most STS(i.e., closest to CPA). This orientation is predominantly based oncomparative mapping data, which places Pax-4 proximal and OB distalwithin the syntenic block present in mouse and human DNA (Zhang et al.1994, supra). The OB gene maps near the telomeric end of the contig,based on the placement of the OB-specific STS (sWSS2619).

While the contig shown in FIG. 35 was deduced by SEGMAP withoutconsideration of YAC sizes (thereby displaying STSs equidistant from oneanother), a similar analysis of the data by SEGMAP that accounted forYAC sizes indicated that the total size of the region covered by thecontig is just over 2 Mb (data not shown). Thus, while all 8 of themicrosatellite-specific STSs (Table 4) are contained within a genomicinterval spanning roughly 2 Mb, the 3 closest to the telomeric end ofthe contig (sWSS1392, sWSS1148, and sWSS2367) are particularly close tothe OB gene itself (perhaps within an interval as small as approximately500 kb). In fact, all 3 of the latter STSs are present in at least 1 ofthe human OB-containing YACs. Of note, the interval between human Pax4(sWSS808) and OB (sWSS2619) is estimated to be approximately 400 kb,whereas this region was predicted to span approximately 1 Mb in mouse(Zhang et al., 1994, supra). Finally, 3 of the YACs within the contig(yWSS691, yWSS999, and yWSS2935) have also been analyzed by FISH, andeach was found to hybridize exclusively to 7q31.3. One of these YACs(yWSS691) contains the OB-specific STS, while the other 2 clones containthe Pax4-specific STS. The latter results are generally consistent withthe previous cytogenetic assignment of human Par4 to 7q32 (Tamura et al.1994, supra). Based on these data, the human OB gene can be assigned tocytogenetic band 7q31.3.

EXAMPLE 11: Human OB Polypeptide is Biologically Active in Mice

Groups of 10 ob/ob mice were treated by i.p. injection with 10 μg/g/dayrecombinant (bacterial) human and murine OB polypeptide or saline. Afterfour days, the group receiving saline gained 0.3 g. The group receivingmurine OB lost 3.2 g. The group receiving human OB lost 2 g (p<0.01compared to saline controls). These groups were also tested for foodintake. The data for food intake are shown in Table 5; the data for bodymass are shown in Table 6.

                                      TABLE 5                                     __________________________________________________________________________    Food intake/day (g) of treated ob/ob mice (value ± S. Dev)                 Treatment                                                                           Day 0                                                                              Day 1                                                                             Day 2                                                                             Day 3                                                                             Day 4                                                                             Day 5                                                                             Day 6                                                                             Day 7                                      __________________________________________________________________________    saline                                                                              13.4 ± 2.6                                                                      12.8                                                                              12.8                                                                              13.1                                                                              14.0                                                                              12.3                                                                              12.4                                                                              8.3                                        murine OB                                                                           14.9 3.7 4.4 5.1 8.9 8.1 8.7 3.5                                        human OB                                                                            14.3 10.3                                                                              8.7 7.0 8.9 5.3 3.8 13.0                                       __________________________________________________________________________

                  TABLE 6                                                         ______________________________________                                        Body weight and weight change in treated ob/ob mice (value ± S.Dev)                                  Percent       Percent                                       Body     Body     change Body   Change                                        Weight   Weight   (Day   Weight (Day 0                                Treatment                                                                             (Day 0)  (Day 4)  0 to 4)                                                                              (Day 6)                                                                              to 6)                                 ______________________________________                                        saline  39.9 ± 1.8                                                                          40.7 ± 1.6                                                                           0.8 ± 0.5                                                                        41.1 ± 2.2                                                                         1.2 ± 1.1                         murine OB                                                                             39.5 ± 2.1                                                                          36.2 ± 2.0                                                                          -3.3 ± 1.2                                                                        36.3 ± 2.2                                                                        -3.1 ± 1.2                         human OB                                                                              39.5 ± 2.0                                                                          37.6 ± 1.7                                                                          -2.0 ± 1.0                                                                        36.1 ± 1.3                                                                        -3.5 ± 1.3                         ______________________________________                                    

These data demonstrate that human OB is biologically active in mice.

EXAMPLE 12: A High Dose of OB Affects Wild-type Mice

Wild-type mice (C57B16J +/?) were treated with 10 μg/day i.p. ofrecombinant murine OB, and body mass was measured every four days. Theresults are shown in Table 7.

                  TABLE 7                                                         ______________________________________                                        Body mass (g) of normal mice receiving OB                                     Treatment                                                                             Day 0    Day 4     Day 8  Day 12 Day 16                               ______________________________________                                        saline  22.6 ± 1.4                                                                          22.2 ± 1.2                                                                           22.5 ± 1.3                                                                        23     22.5                                 murine OB                                                                             22.4 ± 1.5                                                                          20.6 ± 1.5                                                                           20.8 ± 1.3                                                                        20.8   21.8                                 ______________________________________                                    

These data demonstrate that OB affects the body mass of wild-type aswell as obese (ob/ob) mice, albeit to a much smaller degree.

EXAMPLE 13: OB Polypeptide Administered By Continuous Pump Infusion

This example demonstrates that continuous infusion of OB polypeptideresults in weight loss in normal mice. Normal (non-obese) mice wereadministered murine OB polypeptide via osmotic pump infusion. A dosageof 0.5 mg protein/kg body weight/day resulted in a 4.62% loss (+/-1.34%)from baseline weight by the sixth day of infusion.

Materials and Methods

Animals.

Wild type (+/+) C57B16 mice were used in this Example. Mice weresingle-housed, and maintained under humane conditions. The age of themice at the initial time point was 8 weeks, and the animals were weightstabilized. Ten mice were used for each cohort (vehicle vs. protein).

Feeding and weight measurement.

Mice were given ground rodent chow (PMI Feeds, Inc.) in powdered foodfeeders (Allentown Caging and Equipment), which allowed a more accurateand sensitive measurement of food intake than use of regular block chow.Weight was measured at the same time each day (2:00 p.m.), for a periodof 6 days. Body weight on the day prior to infusion was defined asbaseline weight.

Cloning of Murine OB DNA

The cloning of the murine OB DNA for expression in E. coli was performedas follows. The DNA sequence as deduced from the published peptidesequence that appeared in (Zhang et al., 1994, supra, i.e., Example 1,supra) was reverse translated using E. coli optimal codons. The terminalcloning sites were XbaI to BamHI. A ribosomal binding enhancer and astrong ribosomal binding site were included in front of the codingregion. The duplex DNA sequence was synthesized using standardtechniques. Correct clones were confirmed by demonstrating expression ofthe recombinant protein and presence of the correct OB DNA sequence inthe resident plasmid. The amino acid sequence (and DNA sequence) is asfollows:

Recombinant murine met OB (double stranded) DNA and amino acid sequence.(Seq. ID. NOS: 94 and 95):

    TCTAGATTTGAGTTTTAACTTTTAGAAGGAGGAATAACATATGGTACCGATCCAGAAAGT                         9                                                                      -+---------+---------+---------+---------+--------+--------                   68                                                                            AGATCTAAACTCAAAATTGAAAATCTTCCTCCTTATTGTATACCATGGCTAGGTCTTTCA                                                                    M  V  P  I  Q               K  V                                                                          TCAGGACGACACCAAAACCTTAATTAAAACGATCGTTACGCGTATCAACGACATCAGTCA                        69                                                                      -+---------+---------+---------+---------+--------+--------                   128                                                                           AGTCCTGCTGTGGTTTTGGAATTAATTTTGCTAGCAATGCGCATAGTTGCTGTAGTCAGT                            Q  D  D  T  K  T  L  I  K  T  I  V  T  R  I  N  D  I                S  H                                                                          CACCCAGTCGGTCTCCGCTAAACAGCGTGTTACCGGTCTGGACTTCATCCCGGGTCTGCA                       129                                                                      -+---------+---------+---------+---------+--------+--------                   188                                                                           GTGGGTCAGCCAGAGGCGATTTGTCGCACAATGGCCAGACCTGAAGTAGGGCCCAGACGT                            T  Q  S  V  S  A  K  Q  R  V  T  G  L  D  F  I  P  G                L  H                                                                          CCCGATCCTAAGCTTGTCCAAAATGGACCAGACCCTGGCTGTATACCAGCAGGTGTTAAC                       189                                                                      -+---------+---------+---------+---------+--------+--------                   248                                                                           GGGCTAGGATTCGAACAGGTTTTACCTGGTCTGGGACCGACATATGGTCGTCCACAATTG                            P  I  L  S  L  S  K  M  D  Q  T  L  A  V  Y  Q  Q  V                L  T                                                                          CTCCCTGCCGTCCCAGAACGTTCTTCAGATCGCTAACGACCTCGAGAACCTTCGCGACCT                       249                                                                      -+---------+---------+---------+---------+--------+--------                   308                                                                           GAGGGACGGCAGGGTCTTGCAAGAAGTCTAGCGATTGCTGGAGCTCTTGGAAGCGCTGGA                            S  L  P  S  Q  N  V  L  Q  I  A  N  D  L  E  N  L  R                D  L                                                                          GCTGCACCTGCTGGCATTCTCCAAATCCTGCTCCCTGCCGCAGACCTCAGGTCTTCAGAA                       309                                                                      -+---------+---------+---------+---------+--------+--------                   368                                                                           CGACGTGGACGACCGTAAGAGGTTTAGGACGAGGGACGGCGTCTGGAGTCCAGAAGTCTT                            L  H  L  L  A  F  S  K  S  C  S  L  P  Q  T  S  G  L                Q  K                                                                          ACCGGAATCCCTGGACGGGGTCCTGGAAGCATCCCTGTACAGCACCGAAGTTGTTGCTCT                       369                                                                      -+---------+---------+---------+---------+--------+--------                   428                                                                           TGGCCTTAGGGACCTGCCCCAGGACCTTCGTAGGGACATGTCGTGGCTTCAACAACGAGA                            P  E  S  L  D  G  V  L  E  A  S  L  Y  S  T  E  V  V                A  L                                                                          GTCCCGTCTGCAGGGTTCCCTTCAGGACATCCTTCAGCAGCTGGACGTTTCTCCGGAATG                       429                                                                      -+---------+---------+---------+---------+--------+--------                   488                                                                           CAGGGCAGACGTCCCAAGGGAAGTCCTGTAGGAAGTCGTCGACCTGCAAAGAGGCCTTAC                            S  R  L  Q  G  S  L  Q  D  I  L  Q  Q  L  D  V  S  P                E  C                                                                                   TTAATGGATCC                                                               489 -+---------                                                                   AATTACCTAGG                                                      

Expression Vector and Host Strain. The plasmid expression vector usedwas pCFM1656, American Type Culture Collection (ATCC) Accession No.69576. The above DNA was ligated into the expression vector pCFM1656,which had been linearized with XbaI and BamHI, and transformed into theE. coli host strain, FM5. E. coli FM5 cells were derived at Amgen Inc.,Thousand Oaks, Calif. from E. coli K-12 strain [Bachmann et al.,Bacteriol. Rev., 40:116-167 (1976)] and contain the integrated lambdaphage repressor gene, cI₈₅₇ [Sussman et al., C.R. Acad. Sci.,254:1517-1579 (1962)]. Vector production, cell transformation, andcolony selection were performed by standard methods. E.g., Sambrook etal., 1989, supra. Host cells were grown in LB media.

Administration of Protein or Vehicle

Recombinant murine OB polypeptide was used for the present experiments,generally at a concentration of about 0.9 mg/ml phosphate bufferedsaline, pH 7.4. The amino acid sequence (and DNA sequence) used is setout immediately above.

Protein or vehicle (phosphate buffered saline, pH 7.4) were administeredby osmotic pump infusion. Alzet osmotic minipumps (Alza, Palo Alto,Calif., model no. 1007D) were surgically placed in each mice in asubcutaneous pocket in the subscapular area. The pumps were calibratedto administer 0.5 ml protein in solution per hour for a dosage of 0.5 mgprotein/kg body weight/day. Control animals were infused with phosphatebuffered saline (pH 7.4) via an Alzet osmotic minipump.

Fermentation Process. A three-phase fermentation protocol known as afed-batch process was used. Media compositions are set forth below.

Batch. A nitrogen and phosphate source were sterilized (by raising thetemperature to 122° C. for 35 minutes, 18-20 psi) in the fermentationvessel (Biolafitte, 12 liter capacity). Upon cooling, carbon, magnesium,vitamins, and trace metal sources were added aseptically. An overnightculture (16 hours or more) of the above recombinant murineprotein-producing bacteria of 500 ml (grown in LB broth) was added tothe fermentor.

Feed I. Upon reaching between 4.0-6.0 O.D 600, Feed I was added tocultures. The glucose was added at a limiting rate in order to controlthe growth rate (μ). An automated system (called the DistributiveControl System) was programmed to control the growth rate at 0.15generations hr⁻.

Feed II. When the O.D. reached 30, the temperature was slowly increasedto 42° C. and the feed was changed to Feed II, described below. Thefermentation was then allowed to continue for 10 hours with samplingevery 2 hours. After 10 hours, the contents of the fermentor werechilled to below 20° C. and harvested by centrifugation.

Media Composition:

    ______________________________________                                        Batch:      10 g/L      Yeast extract                                                     5.25 g/L    (NH.sub.4).sub.2 SO.sub.4                                         3.5 g/L     K.sub.2 HPO.sub.4                                                 4.0 g/L     KH.sub.2 PO.sub.4                                                 5.0 g/L     Glucose                                                           1.0 g/L     MgSO.sub.4- 7H.sub.2 O                                            2.0 ml/L    Vitamin Solution                                                  2.0 ml/L    Trace Metal Solution                                              1.0 ml/L    P2000 Antifoam                                        Feed I:     50 g/L      Bacto-tryptone                                                    50 g/L      Yeast extract                                                     450 g/L     Glucose                                                           8.75 g/L    MgSO.sub.4- 7H.sub.2 O                                            10 ml/L     Vitamin Solution                                                  10 ml/L     Trace Metal Solution                                  Feed II:    200 g/L     Bacto-tryptone                                                    100 g/L     Yeast extract                                                     110 g/L     Glucose                                               ______________________________________                                    

Vitamin Solution (Batch, Feed 1): 0.5 g biotin, 0.4 g folic acid, and4.2 g riboflavin, were dissolved in 450 ml H₂ O and 3 ml 10 N NaOH, andbrought to 500 ml with H₂ O. Fourteen grams of pyridoxine-HCl and 61grams of niacin were dissolved 150 ml H₂ O and 50 ml 10N NaOH, andbrought to 250 ml with H₂ O. Fifty-four grams of pantothenic acid weredissolved in 200 ml H₂ O, and brought to 250 ml. The three solutionswere combined and brought to 10 liters total volume.

Trace Metal Solution (Batch, Feed I):

Ferric Chloride (FeCl₃.6H₂ O): 27 g/L

Zinc Chloride (ZnCl₂.4H₂ O): 2 g/L

Cobalt Chloride (CoCl₂.6H₂ O): 2 g/L

Sodium Molybdate (NaMoO₄.2H₂ O): 2 g/L

Calcium Chloride (CaCl₂.2H₂ O): 1 g/L

Cupric Sulfate (CuSO₄.5H₂ O): 1.9 g/L

Boric Acid (H₃ BO₃): 0.5 g/L

Manganese Chloride (MnCl₂.4H₂ O): 1.6 g/L

Sodium Citrate dihydrate: 73.5 g/L

Purification Process for Murine OB Polypeptide

Purification was accomplished by the following steps (unless otherwisenoted, the following steps were performed at 4° C.):

1. Cell paste. E. coli cell paste was suspended in 5 times volume of 7mM of EDTA, pH 7.0. The cells in the EDTA were further broken by twopasses through a microfluidizer. The broken cells were centrifuged at4.2k rpm for 1 hour in a Beckman JB-6 centrifuge with a J5-4.2 rotor.

2. Inclusion body wash #1. The supernatant from above was removed, andthe pellet was resuspended with 5 times volume of 7 mM EDTA, pH 7.0, andhomogenized. This mixture was centrifuged as in step 1.

3. Inclusion body wash #2. The supernatant from above was removed, andthe pellet was resuspended in ten times volume of 20 mM Tris, pH 8.5, 10MM DTT, and 1% deoxycholate, and homogenized. This mixture wascentrifuged as in step 1.

4. Inclusion body wash #3. The supernatant from above was removed andthe pellet was resuspended in ten times volume of distilled water, andhomogenized. This mixture was centrifuged as in step 1.

5. Refolding. The pellet was refolded with 15 volumes of 10 mM HEPES, pH8.5, 1% sodium sarcosine (N-lauryl sarcosine), at room temperature.After 60 minutes, the solution was made to be 60 mM copper sulfate, andthen stirred overnight.

6. Removal of sarcosine. The refolding mixture was diluted with fivevolumes of 10 mM Tris buffer, pH 7.5, and centrifuged as in step 1. Thesupernatant was collected, and mixed with agitation for one hour withDowex 1-X4 resin, 20-50 mesh, chloride form (at 0.066% total volume ofdiluted refolding mix). This mixture was poured into a column and theeluant was collected. Removal of sarcosine was ascertained by HPLC.

7. Acid precipitation. The eluant from the previous step was collected,and the pH adjusted to pH 5.5, and incubated for 30 minutes at roomtemperature. This mixture was centrifuged as in step 1.

8. Cation exchange chromatography. The pH of the supernatant from theprevious step was adjusted to pH 4.2, and loaded on CM Sepharose FastFlow. Twenty column volumes of salt gradient were used at 20 mM NaOAc,pH 4.2, 0 M to 1.0 M NaCl.

9. HIC chromatography. The CM Sepharose pool of peak fractions(ascertained from ultraviolet analysis) from the above step was adjustedto be 0.2 M ammonium sulfate. A 20 column volume reverse salt gradientwas done at 5 mM NaOAc, pH 4.2, with 0.4 M to 0 M ammonium sulfate. Thismaterial was concentrated and diafiltered into PBS.

Results

Presented below are the percent (%) differences from baseline weight inC57B16J mice (8wks old):

                  TABLE 8                                                         ______________________________________                                        Weight Loss Upon Continuous Infusion                                                                     Recombinant OB                                     Time (days)  Vehicle (PBS) polypeptide                                        ______________________________________                                        Days 1-2     3.24 +/- 1.13  1.68 +/- 1.4                                      Days 3-4      4.3 +/- .97  -2.12 +/- .79                                      Days 5-6     4.64 +/- .96  -4.62 +/- 1.3                                      ______________________________________                                    

As can be seen, at the end of a 6 day continuous infusion regime,animals receiving the OB polypeptide lost over 4% of their body weight,as compared to baseline. This is a substantially more rapid weight lossthan has been observed with intraperitoneal (i.p.) injection. Weightloss of only 2.6-3.0% was seen at the end of a 32-day injection period,in wild-type (normal) mice, with daily i.p. injections of recombinantmurine OB polypeptide at a 10 mg/kg dose, and had not been more than 4%at any time during the dosing schedule (data not shown). The presentdata indicate that with continuous infusion, a 20-fold lower dosage (0.5mg/kg vs. 10 mg/kg) achieves more weight loss in a shorter time period.

The results seen here are statistically significant, e.g., <4.62% withp<0.0001.

EXAMPLE 14: Cloning and Expression of a Recombinant Human OB Polypeptide

This example provides compositions and methods for preparation of arecombinant human version of the OB polypeptide.

The human version of OB DNA was constructed from the murine OB DNA, asin Example 13, above, by replacing the region between the AuIu and BamHIsites with duplex DNA (made from synthetic oligonucleotides) in which 20codon substitutions had been designed. The MluI site is shown under thesolid line in the sequence below. This DNA was put into the pCFM 1656vector (ATCC Accession No. 69576), in the same fashion as therecombinant murine protein, as described above.

Recombinant human met OB (Double Stranded) DNA and amino acid sequence(Seq. ID. Nos. 97 and 98)

    CATATGGTACCGATCCAGAAAGTTCAGGACGACACCAAAACCTTAATTAAAACGATCGTT                         1                                                                      ---------+---------+---------+---------+----------+---------+                 60                                                                            GTATACCATGGCTAGGTCTTTCAAGTCCTGCTGTGGTTTTGGAATTAATTTTGCTAGCAA                              M  V  P  I  Q  K  V  Q  D  D  T  K  T  L  I  K  T                 I  V                                                                          ACGCGTATCAACGACATCAGTCACACCCAGTCGGTGAGCTCTAAACAGCGTGTTACAGGC                        61                                                                      ---------+---------+---------+---------+----------+---------+                 120                                                                           TGCGCATAGTTGCTGTAGTCAGTGTGGGTCAGCCACTCGAGATTTGTCGCACAATGTCCG                           T  R  I  N  D  I  S  H  T  Q  S  V  S  S  K  Q  R  V                 T  G                                                                          CTGGACTTCATCCCGGGTCTGCACCCGATCCTGACCTTGTCCAAAATGGACCAGACCCTG                       121                                                                      ---------+---------+---------+---------+----------+---------+                 180                                                                           GACCTGAAGTAGGGCCCAGACGTGGGCTAGGACTGGAACAGGTTTTACCTGGTCTGGGAC                           L  D  F  I  P  G  L  H  P  I  L  T  L  S  K  M  D   Q                T  L                                                                          GCTGTATACCAGCAGATCTTAACCTCCATGCCGTCCCGTAACGTTCTTCAGATCTCTAAC                       181                                                                      ---------+---------+---------+---------+----------+---------+                 240                                                                           CGACATATGGTCGTCTAGAATTGGAGGTACGGCAGGGCATTGCAAGAAGTCTAGAGATTG                           A  V  Y  Q  Q  I  L  T  S  M  P  S  R  N  V  L  Q  I                 S  N                                                                          GACCTCGAGAACCTTCGCGACCTGCTGCACGTGCTGGCATTCTCCAAATCCTGCCACCTG                       241                                                                      ---------+---------+---------+---------+----------+---------+                 300                                                                           CTGGAGCTCTTGGAAGCGCTGGACGACGTGCACGACCGTAAGAGGTTTAGGACGGTGGAC                           D  L  E  N  L  R  D  L  L  H  V  L  A  F  S  K  S  C                 H  L                                                                          CCATGGGCTTCAGGTCTTGAGACTCTGGACTCTCTGGGCGGGGTCCTGGAAGCATCCGGT                       301                                                                      ---------+---------+---------+---------+----------+---------+                 360                                                                           GGTACCCGAAGTCCAGAACTCTGAGACCTGAGAGACCCGCCCCAGGACCTTCGTAGGCCA                           P  W  A  S   G   L  E  T  L  D  S  L   G  G  V  L  E   A             S  G                                                                          TACAGCACCGAAGTTGTTGCTCTGTCCCGTCTGCAGGGTTCCCTTCAGGACATGCTTTGG                       361                                                                      ---------+---------+---------+---------+----------+---------+                 420                                                                           ATGTCGTGGCTTCAACAACGAGACAGGGCAGACGTCCCAAGGGAAGTCCTGTACGAAACC                           Y  S  T  E  V  V  A  L  S  R  L  Q  G  S  L  Q  D   M                L  W                                                                                   CAGCTGGACCTGTCTCCGGGTTGTTAATGGATCC                                        421 ----------+---------+---------+----  454                                      GTCGACCTGGACAGAGGCCCAACAATTACCTAGG                                            Q  L  D  L  S  P  G  C   *                                       

Fermentation.

Fermentation of the above host cells to produce recombinant human OBpolypeptide was accomplished using the conditions and compositions asdescribed above for recombinant murine material. The results wereanalyzed for yield (grams/liter), pre-purification, of the recombinanthuman OB material (and minor amounts of bacterial protein), andcorrelated to analyze bacterial expression:

                  TABLE 9                                                         ______________________________________                                        Analysis of Human OB Polypeptide Expression                                              OD            Yield  Expression                                    Timepoint  (@600 nm)     (g/L)  (mg/OD·L)                            ______________________________________                                        Ind. + 2 hrs.                                                                            47             1.91   41                                           Ind. + 4 hrs.                                                                            79             9.48  120                                           Ind. + 6 hrs.                                                                            95            13.01  137                                           Ind. + 8 hrs.                                                                            94            13.24  141                                            Ind. + 10 hrs.                                                                          98            14.65  149                                           ______________________________________                                         abbreviations: Ind. + .sub.--  hours means the hours after induction of       protein expression, as described in Example 13 for the recombinant murine     material using pCFM 1656                                                      O.D.: optical density, as measured by spectrophotometer milligrams per        O.D. unit per liter mg/O.D.·L: expression in terms of mg of          protein per O.D. unit per liter.                                         

Purification of the recombinant human OB polypeptide.

Recombinant human protein may be purified using methods similar to thoseused for purification of recombinant murine protein, as in Example 13,above. For preparation of recombinant human OB polypeptide, step 8 wasperformed by adjusting the pH of the supernatant from step 7 to pH 5. 0,and loading this on to a CM Sepharose fast flow column. The 20 columnvolume salt gradient was performed at 20 mM NaOAc, pH 5.5, 0 to 0.5 MNaCl. Step 9 was performed by diluting the CM Sepharose pool four-foldwith water, and adjusting the pH to 7.5. This mixture was made to 0.7 Mammonium sulfate. A twenty column volume reverse salt gradient was doneat 5 mM NaOAc, pH 5.5, 0 to 0.2 M ammonium sulfate. Otherwise, the abovesteps were identical.

EXAMPLE 15: Dose Response Studies

An additional study demonstrated that there was a dose response tocontinuous administration of OB protein. In this study, wild-type mice(non-obese, CD-1 ice, weighing 35-40 g) were administered recombinantmurine OB protein using methods similar to Examples 12 and 13. Theresults were as follows (with % body weight lost as compared tobaseline, measured as above):

                  TABLE 10                                                        ______________________________________                                        Dose Response With Continuous Administration                                                       % REDUCTION IN BODY                                      DOSE         TIME    WEIGHT                                                   ______________________________________                                        0.03 mg/kg/day                                                                             Day 2   3.5%                                                     1 mg/kg/day  Day 2   7.5%                                                     1 mg/kg/day  Day 4   14%                                                      ______________________________________                                    

As can be seen, increasing the dose from 0.03 mg/kg/day to 1 mg/kg/dayincreased the weight lost from 3.5% to 7.5%. It is also noteworthy thatat day 14, the 1 mg/kg/day dosage resulted in a 14% reduction in bodyweight.

EXAMPLE 16: Effects of Leptin on Body Composition of ob/ob Mice

C57B1/6J ob/ob 16 week old mice were treated with 5 μg/g/day of murineleptin, vehicle, or received no treatment for 33 days. In a secondexperiment, 7 week old ob/ob mice were treated with 10 μg/g/day of humanleptin, murine leptin, or vehicle for 12 days. The mice were sacrificedand total body weight, body composition, insulin levels, and glucoselevels were evaluated. The data from these experiments are reported inTable 11.

                                      TABLE 11                                    __________________________________________________________________________    Body Weight, Composition, Insulin Levels, and Glucose Levels of Treated       Mice                                                                          Treatment Group                                                                         16 Week Old Mice, 5 μg/g/day leptin                                                             7 Week Old Mice, 10 μg/g/day leptin         Treatment Murine leptin                                                                        Vehicle                                                                              Control                                                                              Human Leptin                                                                         Murine Leptin                                                                        Vehicle                          __________________________________________________________________________    Total Body Weight                                                                       31.90 ± 2.8                                                                       64.10 ± 4.5                                                                       67.50 ± 6.2                                                                       31.00 ± 1.3                                                                       33.40 ± 2.4                                                                       42.70 ± 1.5                   Fat  Total (g)                                                                           9.10 ± 1.7                                                                       38.30 ± 4.0                                                                       40.87 ± 6.1                                             %    28.40 ± 3.4                                                                       59.70 ± 2.1                                                                       60.34 ± 3.7                                        Lean Mass                                                                          Total (g)                                                                           6.80 ± 1.0                                                                        7.60 ± 0.4                                                                        7.73 ± 0.5                                             %    21.30 ± 1.7                                                                       11.90 ± 1.2                                                                       11.57 ± 1.6                                        Water                                                                              Total (g)                                                                          16.00 ± 0.8                                                                       18.20 ± 0.7                                                                       18.90 ± 1.0                                             %    50.30 ± 4.2                                                                       28.40 ± 1.0                                                                       28.10 ± 2.2                                        Insulin (UIU/ml)                                                                        <2.0   <2.0   <2.0   <2.0   <2.0   21.4                             Glucose (mg/dl)                                                                          170.0 ± 20.9                                                                       337 ± 30.3                                                                       317.5 ± 51.0                                                                     258.3 ± 26.8                                                                      320.0 ± 44.0                                                                      789.0 ± 152.1                 __________________________________________________________________________

The body composition data demonstrate the effect of leptin on threecompartments of the body: fat mass, lean body mass, and water mass. Thedata indicate that leptin significantly decreases body fat mass and hasa marginal effect on lean body mass. However, the effects on lean bodymass were not statistically significant. Comparison of the insulin andglucose levels in leptin treated and control (untreated) mice indicatesthat leptin reduces blood sugar and insulin levels, and thus amelioratesthese indicia of diabetes.

EXAMPLE 17: High Dose Effects of Leptin on Wild-type Mice

Lean controls of the ob/ob mice (C57B 1/6J+/?) were injected once a day,i.p., with 10 μg/g murine leptin or vehicle (PBS); body weight and foodintake were measured over the next two weeks. There was a significantdecrease in body weight from day four onward and a significant decreasein food intake for the first week. However, after one week, the levelsof food intake became indistinguishable between both groups of mice. Theanimals were sacrificed at the end of the two weeks and body compositionwas determined. The results of the body composition analysis are shownin Table 12. The data show a decrease in body fat of the animalsreceiving leptin versus the animals receiving PBS.

                                      TABLE 12                                    __________________________________________________________________________    Body Composition and Weight of Wildtype (+/?) Mice                                           FAT     LEAN BODY MASS                                                                          WATER                                        GROUP                                                                             C57B1/6J                                                                           BODY WT                                                                             Total                                                                            %    Total                                                                              %    Total                                                                            %                                         __________________________________________________________________________    Protein                                                                            1   17.3  0.30                                                                             1.71%                                                                              5.00 28.93%                                                                             12.00                                                                            69.36%                                         2   20.5  2.39                                                                             11.65%                                                                             5.41 26.40%                                                                             12.70                                                                            61.95%                                         3   16.9  0.34                                                                             1.99%                                                                              4.76 28.19%                                                                             11.80                                                                            69.82%                                         4   18.3  0.84                                                                             4.62%                                                                              5.16 28.17%                                                                             12.30                                                                            67.21%                                         5   17.7  0.44                                                                             2.51%                                                                              4.96 28.00%                                                                             12.30                                                                            69.49%                                         6   18.7  2.56                                                                             13.72%                                                                             4.84 25.86%                                                                             11.30                                                                            60.43%                                         7   15.7  0.37                                                                             2.38%                                                                              4.53 28.83%                                                                             10.80                                                                            68.79%                                         8   16.4  0.29                                                                             1.79%                                                                              4.51 27.48%                                                                             11.60                                                                            70.73%                                         9   16.5  0.83                                                                             5.05%                                                                              4.67 28.29%                                                                             11.00                                                                            66.67%                                        10   14.9                    10.40                                                                            69.80%                                        Avg. 17.3  0.93                                                                             5.04%                                                                              4.87 27.79%                                                                             11.62                                                                            67.43%                                        Std. Dev.                                                                          1.6   0.90                                                                             4.52%                                                                              0.30  1.05%                                                                             0.74                                                                              3.52%                                    Vehicle                                                                           11   18.8  1.30                                                                             6.93%                                                                              5.00 26.58%                                                                             12.50                                                                            66.49%                                        12   17.6  2.17                                                                             12.34%                                                                             4.53 25.73%                                                                             10.90                                                                            61.93%                                        13   18.0  2.29                                                                             12.74%                                                                             4.61 25.59%                                                                             11.10                                                                            61.67%                                        14   19.6  3.79                                                                             19.34%                                                                             4.61 23.52%                                                                             11.20                                                                            57.14%                                        15   18.6  2.35                                                                             12.65%                                                                             4.75 25.52%                                                                             11.50                                                                            61.83%                                        16   17.3  1.96                                                                             11.32%                                                                             4.54 26.25%                                                                             10.80                                                                            62.43%                                        17   19.3  1.38                                                                             7.12%                                                                              5.02 26.04%                                                                             12.90                                                                            66.84%                                        18   20.6  4.16                                                                             20.19%                                                                             4.94 23.98%                                                                             11.50                                                                            55.83%                                        19   17.7  1.08                                                                             6.13%                                                                              4.72 26.64%                                                                             11.90                                                                            67.23%                                        20   19.5                    12.30                                                                            63.08%                                        Avg. 18.7  2.28                                                                             12.09%                                                                             4.75 25.54%                                                                             11.66                                                                            62.45%                                        Std. Dev.                                                                          1.1   1.07                                                                             5.08%                                                                              0.20  1.10%                                                                             0.72                                                                              3.83%                                    __________________________________________________________________________

A second experiment showed the effects of twice a day i.p. injections of12.5 μg/g of murine leptin on wild-type C57B1/6J mice. There was asignificant decrease in body weight and food intake associated withtwice daily injections of the polypeptide. For this experiment, theanimals were placed in metabolic chambers. Food consisted of a powderedPurina #5001 chow diet. This diet differed from earlier experiments,which used the diet consisting of chow diet, tapioca, and water. Thusthe food used in the metabolic chambers had a higher caloric content,which explains why the amount of food consumed differs from thoseanimals on the water-containing diet.

The following is a list of references related to the above disclosureand particularly to the experimental procedures and discussions.

Bahary et al., Genomics, 11:33-47 (1991).

Bahary et al., in Chromosomal microdissection of midmouse chromosome 4:Mapping of microclones relative to the mouse db gene (1991). Submitted.

Bahary et al., Molecular mapping of mouse chromosomes 4 and 6: Use of aflow-sorted Robertsonian chromosome (1991). Submitted.

Blank et al., Mammalian Genome, 1:s51-s78 (1991).

Bogardus et al., Annals of the New York Academy of Sciences, 630:100-115(1991).

Friedman et al., Mammalian Genome, 1: 130-144 (1991).

Harris, FASEB J., 4:3310-3318 (1990).

Jacobowitz et al., N. Engl. J. Med., 315:96-100 (1986).

Kessey, in Obesity, pp. 144-166, Stunkard ed., Philadelphia, W.B.Sauders Co. (1980).

Kessey et al., Ann. Rev. Psychol., 37:109-133.22 (1986).

Leibel et al., "Genetic variation and nutrition in obesity: Approachesto the molecular genetics of obesity", in Genetic Variation andNutrition, pp. 90-101.1, Simopoulos and Childs eds., S. Karger, Basel(1990).

Siegel et al., Localization of the cystic fibrosis transmembraneconductance regulator to mouse chromosome 6. Cytogenetics Cell Genetics(1991). Submitted.

This invention may be embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allrespects illustrative and not restrictive, the scope of the inventionbeing indicated by the appended claims, and all changes which comewithin the meaning and range of equivalency are intended to be embracedtherein.

This invention may be embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allrespects illustrative and not restrictive, the scope of the inventionbeing indicated by the appended claims, and all changes which comewithin the meaning and range of equivalency are intended to be embracedtherein.

Various references are cited throughout this specification, each ofwhich is incorporated herein by reference in its entirety.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 99                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 2793 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       #ob cDNA  (A) DESCRIPTION: Murine                                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Murine                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 57..560                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 #AGGAAA        56AGCAGCT GCAAGGTGCA AGAAGAAGAA GATCCCAGGG                     #TGG CTT TGG TCC TAT CTG     104GG TTC CTG                                    #Trp Leu Trp Ser Tyr Leu Cys Arg Phe Leu                                      #15                                                                           #GTC CAG GAT GAC ACC AAA     152TC CAG AAA                                    #Val Gln Asp Asp Thr Lys Pro Ile Gln Lys                                      #              30                                                             #AAT GAC ATT TCA CAC ACG     200CC AGG ATC                                    #Asn Asp Ile Ser His Thr Val Thr Arg Ile                                      #          45                                                                 #GGC TTG GAC TTC ATT CCT     248GG GTC ACT                                    #Gly Leu Asp Phe Ile Pro Gln Arg Val Thr                                      #      60                                                                     #ATG GAC CAG ACT CTG GCA     296TG TCC AAG                                    #Met Asp Gln Thr Leu Ala Ser Leu Ser Lys                                      #  80                                                                         #TCC CAA AAT GTG CTG CAG     344GC CTG CCT                                    #Ser Gln Asn Val Leu Gln Thr Ser Leu Pro                                      #95                                                                           #CTC CTC CAT CTG CTG GCC     392TC CGA GAC                                    #Leu Leu His Leu Leu Ala Asn Leu Arg Asp                                      #            110                                                              #AGT GGC CTG CAG AAG CCA     440CT CAG ACC                                    #Ser Gly Leu Gln Lys Pro Leu Pro Gln Thr                                      #        125                                                                  #CTC TAC TCC ACA GAG GTG     488AA GCC TCA                                    #Leu Tyr Ser Thr Glu Val Leu Glu Ala Ser                                      #    140                                                                      #CAG GAC ATT CTT CAA CAG     536GC TCT CTG                                    #Gln Asp Ile Leu Gln Gln Gln Gly Ser Leu                                      #160                                                                          #GCCACCAGGC TCCCAAGA      588C TGA AGTTTCAAAG                                  Leu Asp Val Ser Pro Glu Cys  *                                                                165                                                          #AGCCATGTGC   648AAGAAAC CTTGGCTTCC AGGGGTCTTC AGGAGAAGAG                     #CATGACTCCA   708TCATTTC TCTCCCTCCT GTAGACCACC CATCCAAAGG                     #CAGCCTGCAG   768AAGTTAT CCACACAACT TCATGAGCAC AAGGAGGGGC                     #CCCCTCCATG   828CTAGTTC TTCAGCAAGT AGAGATAAGA GCCATCCCAT                     #GCCCAGGAGA   888GGGTACA TGTTCCTCCG TGGGTACACG CTTCGCTGCG                     #GCACCGTGAA   948TGGGTAG AGCCTTTGGG CTGTCTCAGA GTCTTTGGGA                     #ACTTATTTAT  1008CACAGCT GGAAACTCCC AAGCAGCACA CGATGGAAGC                     #CAGGCTTTGG  1068TATTTTG GATGGATCTG AAGCAAGGCA TCAGCTTTTT                     #GATGGGTCTG  1128TGAGGAA GGCTCCTGGG GTGCTGCTTT CAATCCTATT                     #CAAACAAGAG  1188TAATTTT TGAGTGACTG GAAGGAAGGT TGGGATCTTC                     #ATTGTGACTG  1248CGCTCAA GATTGACCTC TGGTGACTGG TTTTGTTTCT                     #TATTATCAAA  1308ACGTTTG CAGCGGCATT GCCGGGAGCA TAGGCTAGGT                     #AGAGGATGTG  1368TGTCAAG TGTAATATGT ATCTATGTGC ACCTGAGGGT                     #GGTAGGCTTT  1428GAAGGAT CCGGAAGTGT TCTCTGAATT ACATATGTGT                     #TGTGAAAAGG  1488GGCATTT TCTTACCTCT GTGGCCACAT AGTGTGGCTT                     #GGAGGGGCTA  1548CTCTTTC CGGAACATTT GGAGTGTACC AGGCACCCTT                     #CCACATTTGA  1608TTTGTTG GCATATTGCT GAGCTCAGGG AGTGAGGGCC                     #CAGGGTTGAT  1668AAGAAAA GGGTCCCTGG TGTAGATCTC CAAGGTTGTC                     #TCTCATCTGA  1728TCTTAAG CAGGTAGACG TTTGCATGCC AATATGTGGT                     #TTGTTCCAGT  1788AGTAGAA CCCTGTCTCC CACCCATTCT GTGGGGAGTT                     #CTGAGGAAGT  1848CACTTAG CAGATGGTCC TGAGCCCTGG GCCAGCACTG                     #AACAAAGGGG  1908GCCAGGC TGCCAGAATT GCCCTTCGGG CTGGAGGATG                     #GCAGATCAGT  1968ATCACCC CTGCACCCTA TGTCACCATC AAACTGGGGG                     #GTGCTCAGCT  2028ATGGAAA GCAATACACT TTAAGACTGA GCACAGTTTC                     #GAGGCTCATG  2088TGAGCTA GAGAAGCTCA CCACATACAT ATAAAAATCA                     #CGCACCGCTG  2148ACCCTAC TCGCGGCGGT GTACTCCACC ACAGCAGCAC                     #ACAGTGCCCA  2208GTCTTCA ACAGGTGTGA AAGAACCTGA GCTGAGGGTG                     #CATTAGCATC  2268TGCAGTC TATTGCATTT ACATACCGCA TTTCAGGGCA                     #CTATCCCTTA  2328GCACACT GTTGACAATA GGACAAGGGA TAGGGGTTGA                     #CATAGGTATA  2388GGACTAG AAGAGTTTTG GATTTTAGAG TCTTTTCAGG                     #CATGAAGTTC  2448AAAATGA GATATCTTGG GGATGGGGCC CAAGTATAAA                     #CAGTGTTTTA  2508AATACCG TATAGACACT GCTTGAAGTG TAGTTTTATA                     #CATGCCAGCA  2568GCATGAA AGACGTTTTT ACAGCATGAA CCTGTCTACT                     #AGAGATGGTT  2628GGGTTTT GGAGCAGTTT GGATCTTGGG TTTTCTGTTA                     #TCCTCAGCCC  2688AACCATA ATGGCAAACA GGCTGCAGGA CCAGACTGGA                     #AGGTTTTGTG  2748TCCAGCC AGGTCATACC CTGTGGAGGT GAGCGGGATC                     #                2793GGA GGTAGATTTT GGAGGATCTG AGGGC                          - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 167 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             #ob polypeptideESCRIPTION: Murine                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 #Trp Leu Trp Ser Tyr Leueu Cys Arg Phe Leu                                    #15                                                                           #Val Gln Asp Asp Thr Lysal Pro Ile Gln Lys                                    #              30                                                             #Asn Asp Ile Ser His Thrle Val Thr Arg Ile                                    #          45                                                                 #Gly Leu Asp Phe Ile Proys Gln Arg Val Thr                                    #      60                                                                     #Met Asp Gln Thr Leu Alaeu Ser Leu Ser Lys                                    #  80                                                                         #Ser Gln Asn Val Leu Glneu Thr Ser Leu Pro                                    #95                                                                           #Leu Leu His Leu Leu Alalu Asn Leu Arg Asp                                    #            110                                                              #Ser Gly Leu Gln Lys Proer Leu Pro Gln Thr                                    #        125                                                                  #Leu Tyr Ser Thr Glu Valal Leu Glu Ala Ser                                    #    140                                                                      #Gln Asp Ile Leu Gln Glneu Gln Gly Ser Leu                                    #160                                                                          -  Leu Asp Val Ser Pro Glu Cys                                                                 165                                                          - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 700 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                #ob cDNA where N represents anyn                                                            nucleotide                                                      -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 46..546                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 -  NNNGNNGTTG CAAGGCCCAA GAAGCCCANN NTCCTGGGAA GGAAA ATG - # CAT TGG            54                                                                          #Trp            Met His                                                       #                 1                                                           #CCC TAT CTT TTC TAT GTC     102GG CTT TGG                                    #Pro Tyr Leu Phe Tyr Val Leu Trp Leu Trp                                      #       15                                                                    #GAC ACC AAA ACC CTC ATC     150TC CAA GAT                                    #Asp Thr Lys Thr Leu Ile Lys Val Gln Asp                                      #  35                                                                         #TCA CAC ACG CAG TCA GTC     198AT GAC ATT                                    #Ser His Thr Gln Ser Val Ile Asn Asp Ile                                      #50                                                                           #TTC ATT CCT GGG CTC CAC     246GT TTG GAC                                    #Phe Ile Pro Gly Leu His Thr Gly Leu Asp                                      #              65                                                             #ACA CTG GCA GTC TAC CAA     294TG GAC CAG                                    #Thr Leu Ala Val Tyr Gln Lys Met Asp Gln                                      #          80                                                                 #GTG ATC CAA ATA TCC AAC     342CC AGA AAC                                    #Val Ile Gln Ile Ser Asn Pro Ser Arg Asn                                      #      95                                                                     #GTG CTG GCC TTC TCT AAG     390TT CTT CAC                                    #Val Leu Ala Phe Ser Lys Asp Leu Leu His                                      #115                                                                          #GAG ACC TTG GAC AGC CTG     438GT GGC CTG                                    #Glu Thr Leu Asp Ser Leu Ala Ser Gly Leu                                      #                130                                                          #ACA GAG GTG GTG GCC CTG     486GC TAC TCC                                    #Thr Glu Val Val Ala Leu Ser Gly Tyr Ser                                      #            145                                                              #CTG TGG CAG CTG GAC CTC     534AG GAC ATG                                    #Leu Trp Gln Leu Asp Leu Leu Gln Asp Met                                      #        160                                                                  #GGACTNACGT          585CCTT GAAGGTCACT CTTCCTGCAA                             Ser Pro Gly Cys                                                                   165                                                                      #CATGGACACC   645TCTGGTT TCCAGGTATC TCCAGGATTG AAGAGCATTG                     #AAAGG        700TCTGTCA ATTTCCCTGA CTCCTCTAAG CCACTCTTCC                     - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 167 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             #ob polypeptideESCRIPTION: Human                                              -     (vi) ORIGINAL SOURCE: Human                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 #Trp Leu Trp Pro Tyr Leueu Cys Gly Phe Leu                                    #15                                                                           #Val Gln Asp Asp Thr Lysal Pro Ile Gln Lys                                    #              30                                                             #Asn Asp Ile Ser His Thrle Val Thr Arg Ile                                    #          45                                                                 #Gly Leu Asp Phe Ile Proys Gln Lys Val Thr                                    #      60                                                                     #Met Asp Gln Thr Leu Alaeu Thr Leu Ser Lys                                    #  80                                                                         #Ser Arg Asn Val Ile Glneu Thr Ser Met Pro                                    #95                                                                           #Leu Leu His Val Leu Alalu Asn Leu Arg Asp                                    #            110                                                              #Ser Gly Leu Glu Thr Leuis Leu Pro Trp Ala                                    #        125                                                                  #Gly Tyr Ser Thr Glu Valal Leu Glu Ala Ser                                    #    140                                                                      #Gln Asp Met Leu Trp Glneu Gln Gly Ser Leu                                    #160                                                                          -  Leu Asp Leu Ser Pro Gly Cys                                                                 165                                                          - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 166 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             #ob polypeptide lacking Gln at position                                                      49                                                             -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Murine                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #Trp Leu Trp Ser Tyr Leueu Cys Arg Phe Leu                                    #15                                                                           #Val Gln Asp Asp Thr Lysal Pro Ile Gln Lys                                    #              30                                                             #Asn Asp Ile Ser His Thrle Val Thr Arg Ile                                    #          45                                                                 #Leu Asp Phe Ile Pro Glyln Arg Val Thr Gly                                    #      60                                                                     #Asp Gln Thr Leu Ala Valer Leu Ser Lys Met                                    #  80                                                                         #Gln Asn Val Leu Gln Ilehr Ser Leu Pro Ser                                    #95                                                                           #Leu His Leu Leu Ala Phesn Leu Arg Asp Leu                                    #            110                                                              #Gly Leu Gln Lys Pro Glueu Pro Gln Thr Ser                                    #        125                                                                  #Tyr Ser Thr Glu Val Valeu Glu Ala Ser Leu                                    #    140                                                                      #Asp Ile Leu Gln Gln Leuln Gly Ser Leu Gln                                    #160                                                                          -  Asp Val Ser Pro Glu Cys                                                                     165                                                          - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 166 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             #ob polypeptide lacking Gln at position                                                      49                                                             -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 #Trp Leu Trp Pro Tyr Leueu Cys Gly Phe Leu                                    #15                                                                           #Val Gln Asp Asp Thr Lysal Pro Ile Gln Lys                                    #              30                                                             #Asn Asp Ile Ser His Thrle Val Thr Arg Ile                                    #          45                                                                 #Leu Asp Phe Ile Pro Glyln Lys Val Thr Gly                                    #      60                                                                     #Asp Gln Thr Leu Ala Valhr Leu Ser Lys Met                                    #  80                                                                         #Arg Asn Val Ile Gln Ilehr Ser Met Pro Ser                                    #95                                                                           #Leu His Val Leu Ala Phesn Leu Arg Asp Leu                                    #            110                                                              #Gly Leu Glu Thr Leu Aspeu Pro Trp Ala Ser                                    #        125                                                                  #Tyr Ser Thr Glu Val Valeu Glu Ala Ser Gly                                    #    140                                                                      #Asp Met Leu Trp Gln Leuln Gly Ser Leu Gln                                    #160                                                                          -  Asp Leu Ser Pro Gly Cys                                                                     165                                                          - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 176 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                                 (A) DESCRIPTION: exon 2 - #G7                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 #TGTCGGGTCC    60AAGATCC CAGGGCAGGA AAATGTGCTG GAGACCCCTG                     #GTCCAGGATG   120CTATCTG TCTTATGTNC AAGCAGTGCC TATCCAGAAA                     #CACACG       176CATCAAG ACCATTGTCA NCAGGATCAC TGANATTTCA                     - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        # primer for exon 2G7TION: PCR 5                                              -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 #  18              GTG                                                        - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        # primer for exon 2G7TION: PCR 3                                              -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: YES                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 #                 22GATA GG                                                   - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 23 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             #N-terminal signal peptide putative                                           -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                #Trp Leu Trp Ser Tyr Leueu Cys Arg Phe Leu                                    #                 15                                                          -  Ser Tyr Val Gln Ala Val Pro                                                             20                                                               - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 287 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                              -     (ii) MOLECULE TYPE: DNA (plasmid)                                       #expression vectorRIPTION: pET-15b                                            -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: T7 promot - #er                                                 (B) LOCATION: 20..37                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: lac opera - #tor                                                (B) LOCATION: 39..64                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 108..243                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: His-Tag                                                         (B) LOCATION: 123..137                                              -     (ix) FEATURE:                                                           #cleavage siteNAME/KEY: Thrombin                                                        (B) LOCATION: 184..196                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                #CGGATAACAA    60GCGAAAT TAATACGACT CACTATAGGG GAATTGTGAG                     -  TTCCCCTCTA CAAATAATTT TGTTTAACTT TAAGAAGGAG ATATACC A - #TG GGC AGC         116                                                                          #                 Met - # Gly Ser                                             # 1                                                                           #CTG GTG CCG CGC GGC AGC     164GC AGC GGC                                    #Leu Val Pro Arg Gly Ser His Ser Ser Gly                                      #       15                                                                    #GCC CGA AAG GAA GCT GAG     212CT AAC AAA                                    #Ala Arg Lys Glu Ala Glu Ala Ala Asn Lys                                      #  35                                                                         #G CATAACCCCT TGGGGCCTCT     263AA TAA CTA                                     Leu Ala Ala Ala Thr Ala Glu Gln  *                                           #40                                                                           #               287GGTTT TTTG                                                 - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 43 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                #Ser Ser Gly Leu Val Prois His His His His                                    #15                                                                           #Ala Asn Lys Ala Arg Lyseu Glu Asp Pro Ala                                    #              30                                                             #Glnlu Ala Glu Leu Ala Ala Ala Thr Ala Glu                                    #          40                                                                 - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 32 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #5  primer(A) DESCRIPTION: Murine                                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                #          32      TGCCG ATCCAGAAAG TC                                        - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 32 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #3  primer(A) DESCRIPTION: Murine                                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                #          32      CTTGG GAGCCTGGTG GC                                        - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 32 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #5  primer(A) DESCRIPTION: Human                                              -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                #          32      TGCCG ATCCAAAAAG TC                                        - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 32 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #3  primer(A) DESCRIPTION: Human                                              -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: Yes                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                #          32      TACTC CTTGCAGGAA GA                                        - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 11 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                #acceptor site in obPTION: Splice                                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Splice ac - #ceptor site                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                #       11                                                                    - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 16 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                                       (A) DESCRIPTION: ob pep - #tide fragment                            -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Murine                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                #Lys Thr Leu Ile Lys Thral Gln Asp Asp Thr                                    #                 15                                                          - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 15 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                                       (A) DESCRIPTION: ob pep - #tide fragment                            -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Murine                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                #Asp Gln Thr Leu Alaeu Ser Leu Ser Lys Met                                    #                 15                                                          - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 19 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                                       (A) DESCRIPTION: ob pep - #tide fragment                            -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Murine                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                #Gly Leu Gln Lys Pro Glueu Pro Gln Thr Ser                                    #                 15                                                          -  Ser Leu Asp                                                                - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                                       (A) DESCRIPTION: ob pep - #tide fragment                            -      (v) FRAGMENT TYPE: Carboxyl terminal                                   -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Murine                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                #Leu Gln Gln Leu Asp Valer Leu Gln Asp Ile                                    #                 15                                                          -  Ser Pro Glu Cys                                                                         20                                                               - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 414 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       #of the human ob gene includingion                                            #sequence upstream of first exon, coding                                      #of first exon, and 5 region of first intron                                  -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 38..181                                               -     (ix) FEATURE:                                                                     (A) NAME/KEY: 5  reg - #ion of first intron                                   (B) LOCATION: 182..414                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: 5  non - #coding sequence of the human ob gene        from                                                                                         which the - # HOB 1gF DNA primer was generated                           (B) LOCATION: 11..28                                                -     (ix) FEATURE:                                                           #sequence of the human ob gene from                                           #HOB 1gR primer was generated                                                           (B) LOCATION: 241..260                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                -  GGTTGCAAGG CCCAAGAAGC CCATCCTGGG AAGGAAA ATG CAT T - #GG GGA ACC CTG         55                                                                          #      Met His Trp Gly Thr Leu                                                #    5  1                                                                     #TTC TAT GTC CAA GCT GTG     103CC TAT CTT                                    #Phe Tyr Val Gln Ala Val Trp Pro Tyr Leu                                      #             20                                                              #ACC CTC ATC AAG ACA ATT     151AC ACC AAA                                    #Thr Leu Ile Lys Thr Ile Asp Asp Thr Lys                                      #         35                                                                  #GTAAGGAGAG TATGCGGGGA       201CA CAC ACG                                     Val Thr Arg Ile Asn Asp Ile Ser His Thr                                      #     45                                                                      #CAGATAGTCC   261CAGCCAG CCCAGCACTG GCTCCTAGTG GCACTGGACC                     #GAGAAGGATT   321TGAACGC CTCCTGAATG CCAGGCACCT ACTGGAAGCT                     #TGCCTGCTGA   381GGCTCCA CTCTTTCTGG TTGTTTCTTN TGGCCCCCTC                     #        414       NGGTT CTTAATTCCT AAA                                       - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 48 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                                       (A) DESCRIPTION: N-termina - #l portion of the human ob             protein                                                                                      encoded b - #y first exon                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                #Trp Leu Trp Pro Tyr Leueu Cys Gly Phe Leu                                    #                 15                                                          #Val Gln Asp Asp Thr Lysal Pro Ile Gln Lys                                    #             30                                                              #Asn Asp Ile Ser His Thrle Val Thr Arg Ile                                    #         45                                                                  - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 801 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       #of the human ob gene including 3n                                                           region of - # first intron, coding sequence of second          exon,                                                                         #noncoding sequence3                                                          -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 291..648                                              -     (ix) FEATURE:                                                           #first intron NAME/KEY: 3  of                                                           (B) LOCATION: 1..290                                                -     (ix) FEATURE:                                                           #sequence of the human ob gene HOB from                                                      which the - # HOB 2gF primer was generated                               (B) LOCATION: 250..269                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: 3  non - #coding sequence of the human ob gene        from                                                                                         which the - # HOB 2gR DNA primer was generated                           (B) LOCATION: 707..728                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                #AAATTGGCCT    60GAAGAGG CCATGTAAGA GAAAGGAATT GACCTAGGGA                     #AGCTTAGAGG   120AACGGAT GGTGTGGGAA AAGCAGGAAT CTCGGAGACC                     #GGGAGGGTGG   180TGGGTGC AGGANACAAG GGCCTGAGCC AAAGTGGTGA                     #GCTCTGAGAG   240AGAGAAT GACCCTCCAT GCCCACGGGG AAGGCAGAGG                     #CAG TCA      296ATGCTGA GCACTTGTTC TCCCTCTTCC TCCTNCATAG                     #  Gln Ser                                                                    #   1                                                                         #GAC TTC ATT CCT GGG CTC     344CC GGT TTG                                    #Asp Phe Ile Pro Gly Leu Val Thr Gly Leu                                      #          15                                                                 #CAG ACA CTG GCA GTC TAC     392AG ATG GAC                                    #Gln Thr Leu Ala Val Tyr Ser Lys Met Asp                                      #     30                                                                      #AAC GTG ATC CAA ATA TCC     440CT TCC AGA                                    #Asn Val Ile Gln Ile Ser Met Pro Ser Arg                                      # 50                                                                          #CAC GTG CTG GCC TTC TCT     488AT CTT CTT                                    #His Val Leu Ala Phe Ser Arg Asp Leu Leu                                      #                 65                                                          #CTG GAG ACC TTG GAC AGC     536CC AGT GGC                                    #Leu Glu Thr Leu Asp Ser Trp Ala Ser Gly                                      #             80                                                              #TCC ACA GAG GTG GTG GCC     584CA GGC TAC                                    #Ser Thr Glu Val Val Ala Ala Ser Gly Tyr                                      #         95                                                                  #ATG CTG TGG CAG CTG GAC     632TG CAG GAC                                    #Met Leu Trp Gln Leu Asp Ser Leu Gln Asp                                      #   110                                                                       #TCCTGCAAGG ACTACGTTAA   688GCCTTGA AGGTCACTCT                                 Leu Ser Pro Gly Cys                                                           115                                                                          #TGGACACCCC   748GGCTTTC CAGGTATCTC CAGGATTGAA GAGCATTGCA                     #AGG          801TGTCAAT TTCCCTGACT CCTCTAAGCC ACTCTTCCAA                     - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 119 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                                       (A) DESCRIPTION: C-termina - #l portion of the human ob             protein                                                                                      encoded b - #y second exon                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                #Gly Leu Asp Phe Ile Proys Gln Lys Val Thr                                    #                 15                                                          #Met Asp Gln Thr Leu Alaeu Thr Leu Ser Lys                                    #             30                                                              #Ser Arg Asn Val Ile Glneu Thr Ser Met Pro                                    #         45                                                                  #Leu Leu His Val Leu Alalu Asn Leu Arg Asp                                    #     60                                                                      #Ser Gly Leu Glu Thr Leuis Leu Pro Trp Ala                                    # 80                                                                          #Gly Tyr Ser Thr Glu Valal Leu Glu Ala Ser                                    #                 95                                                          #Gln Asp Met Leu Trp Glneu Gln Gly Ser Leu                                    #           110                                                               -  Leu Asp Leu Ser Pro Gly Cys                                                        115                                                                   - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: pichia ye - #ast                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                -  Leu Glu Lys Arg Glu Ala Glu Ala                                              1               5                                                           - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 4 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: pichia ye - #ast                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                -  Glu Ala Glu Ala                                                              1                                                                           - (2) INFORMATION FOR SEQ ID NO:28:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 4 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: Internal                                            -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: pichia ye - #ast                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                -  Leu Glu Lys Arg                                                              1                                                                           - (2) INFORMATION FOR SEQ ID NO:29:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                                  (A) DESCRIPTION: HOB 1g - #F DNA primer generated from the 5                      noncoding seq - #uence of the human ob gene                     -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                #  18              CTG                                                        - (2) INFORMATION FOR SEQ ID NO:30:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                                  (A) DESCRIPTION: HOB 1g - #R DNA primer generated from the          first                                                                         #sequence of the human ob gene                                                -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: YES                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                # 20               GTGCC                                                      - (2) INFORMATION FOR SEQ ID NO:31:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                                  (A) DESCRIPTION: HOB 2g - #F DNA primer generated from the          first                                                                         #sequence of the human ob gene                                                -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                # 20               TTGTT                                                      - (2) INFORMATION FOR SEQ ID NO:32:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                                  (A) DESCRIPTION: HOB 2g - #R DNA primer generated from the 3                      noncoding seq - #uence of the human ob gene                     -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: YES                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                #                 22ACCT GG                                                   - (2) INFORMATION FOR SEQ ID NO:33:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 51 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                              -     (ii) MOLECULE TYPE: DNA                                                 #cloning site DESCRIPTION: pPIC.9                                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                #G             51AGGCTGA AGCTTACGTA GAATTCCCTA GGCCGGCCGG                     - (2) INFORMATION FOR SEQ ID NO:34:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        # primer for amplifying human ob cDNA                                                       sequence                                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                #    40            AGAGT GCCCATCCAA AAAGTCCAAG                                - (2) INFORMATION FOR SEQ ID NO:35:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        # primer for amplifying human ob cDNA                                                       sequence                                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: YES                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                #          31      ACCCA GGGCTGAGGT C                                         - (2) INFORMATION FOR SEQ ID NO:36:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        # primer for amplifying murine ob cDNA                                                       sequence                                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                #    40            AGAGT GCCTATCCAG AAAGTCCAGG                                - (2) INFORMATION FOR SEQ ID NO:37:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        # primer for amplifying murine ob cDNA                                                       sequence                                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: YES                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                #          31      ATTCA GGGCTAACAT C                                         - (2) INFORMATION FOR SEQ ID NO:38:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 4 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                                       (A) DESCRIPTION: tetrapept - #ide at N-terminus of renatured        #protein after thrombin cleavage                                              -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Murine                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                -  Gly Ser His Met                                                              1                                                                           - (2) INFORMATION FOR SEQ ID NO:39:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS1734                                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                # 19               AAGG                                                       - (2) INFORMATION FOR SEQ ID NO:40:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS1734                                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                #  18              CAG                                                        - (2) INFORMATION FOR SEQ ID NO:41:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS494                                                         -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                # 19               TTCC                                                       - (2) INFORMATION FOR SEQ ID NO:42:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS494                                                         -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                #                 22CCTC TC                                                   - (2) INFORMATION FOR SEQ ID NO:43:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS883                                                         -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                # 20               TTGAG                                                      - (2) INFORMATION FOR SEQ ID NO:44:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS883                                                         -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                # 20               TGAGC                                                      - (2) INFORMATION FOR SEQ ID NO:45:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS2359                                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                #  18              CTG                                                        - (2) INFORMATION FOR SEQ ID NO:46:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS2359                                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                # 19               AGTG                                                       - (2) INFORMATION FOR SEQ ID NO:47:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS2336                                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 #ID NO:47:(xi) SEQUENCE DESCRIPTION: SEQ                                      #  18              TAC                                                        - (2) INFORMATION FOR SEQ ID NO:48:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS2336                                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                # 20               GATTG                                                      - (2) INFORMATION FOR SEQ ID NO:49:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS1218                                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                #  18              AAC                                                        - (2) INFORMATION FOR SEQ ID NO:50:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS1218                                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                # 20               ATGCC                                                      - (2) INFORMATION FOR SEQ ID NO:51:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS1402                                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                #  18              TAC                                                        - (2) INFORMATION FOR SEQ ID NO:52:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS1402                                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                #  18              GAC                                                        - (2) INFORMATION FOR SEQ ID NO:53:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS999                                                         -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                #  18              TTC                                                        - (2) INFORMATION FOR SEQ ID NO:54:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS999                                                         -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                # 19               ACCC                                                       - (2) INFORMATION FOR SEQ ID NO:55:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS1751                                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                #                 22CAGA AG                                                   - (2) INFORMATION FOR SEQ ID NO:56:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS1751                                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                # 20               TACAG                                                      - (2) INFORMATION FOR SEQ ID NO:57:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS1174                                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                #  18              CAC                                                        - (2) INFORMATION FOR SEQ ID NO:58:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS1174                                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                #  18              GAG                                                        - (2) INFORMATION FOR SEQ ID NO:59:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS2061                                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                # 19               ATCC                                                       - (2) INFORMATION FOR SEQ ID NO:60:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS2061                                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                # 20               TATAG                                                      - (2) INFORMATION FOR SEQ ID NO:61:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS2588                                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                #  18              ATC                                                        - (2) INFORMATION FOR SEQ ID NO:62:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS2588                                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                #  18              ACC                                                        - (2) INFORMATION FOR SEQ ID NO:63:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS808                                                         -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                #                24ATCCT AGGA                                                 - (2) INFORMATION FOR SEQ ID NO:64:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS808                                                         -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                                #                23GGCTG GAC                                                  - (2) INFORMATION FOR SEQ ID NO:65:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                          sWSS1392                                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                                #  18              ATC                                                        - (2) INFORMATION FOR SEQ ID NO:66:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS1392                                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                                #  18              TTC                                                        - (2) INFORMATION FOR SEQ ID NO:67:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS1148                                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                                #  18              AAG                                                        - (2) INFORMATION FOR SEQ ID NO:68:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS1148                                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                                # 19               GTAG                                                       - (2) INFORMATION FOR SEQ ID NO:69:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS1529                                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                                #  18              GTC                                                        - (2) INFORMATION FOR SEQ ID NO:70:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS1529                                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                                #  18              TTG                                                        - (2) INFORMATION FOR SEQ ID NO:71:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS2619                                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:                                #21                CTCTG G                                                    - (2) INFORMATION FOR SEQ ID NO:72:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS2619                                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:                                # 20               AGGGA                                                      - (2) INFORMATION FOR SEQ ID NO:73:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS404                                                         -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:                                #  18              AAG                                                        - (2) INFORMATION FOR SEQ ID NO:74:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS404                                                         -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:                                #  18              GCC                                                        - (2) INFORMATION FOR SEQ ID NO:75:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS2367                                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:                                #  18              TTG                                                        - (2) INFORMATION FOR SEQ ID NO:76:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #tagged-site specific PCR primernce                                                         sWSS2367                                                        -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:                                #  18              CTA                                                        - (2) INFORMATION FOR SEQ ID NO:77:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #UT528    (A) DESCRIPTION: Marker                                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:                                # 20               TTGAG                                                      - (2) INFORMATION FOR SEQ ID NO:78:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #UT528    (A) DESCRIPTION: Marker                                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:                                # 20               TGAGC                                                      - (2) INFORMATION FOR SEQ ID NO:79:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #AFMa065zg9A) DESCRIPTION: Marker                                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:                                # 20               AGCCT                                                      - (2) INFORMATION FOR SEQ ID NO:80:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #AFMa065zg9A) DESCRIPTION: Marker                                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:                                # 19               GATT                                                       - (2) INFORMATION FOR SEQ ID NO:81:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #AFMa125wh1A) DESCRIPTION: Marker                                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:                                # 19               ATCC                                                       - (2) INFORMATION FOR SEQ ID NO:82:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #AFMa125wh1A) DESCRIPTION: Marker                                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:                                # 20               TCAAA                                                      - (2) INFORMATION FOR SEQ ID NO:83:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #AFM309yf10A) DESCRIPTION: Marker                                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:                                # 20               TTTTT                                                      - (2) INFORMATION FOR SEQ ID NO:84:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #AFM309yf10A) DESCRIPTION: Marker                                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:                                # 20               AGACA                                                      - (2) INFORMATION FOR SEQ ID NO:85:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 16 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #AFM218xf10A) DESCRIPTION: Marker                                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:                                #    16            A                                                          - (2) INFORMATION FOR SEQ ID NO:86:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #AFM218xf10A) DESCRIPTION: Marker                                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:                                # 20               TGTCA                                                      - (2) INFORMATION FOR SEQ ID NO:87:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 16 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #AFM206xc1(A) DESCRIPTION: Marker                                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:                                #    16            C                                                          - (2) INFORMATION FOR SEQ ID NO:88:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #AFM206xc1(A) DESCRIPTION: Marker                                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:                                # 20               TCAGT                                                      - (2) INFORMATION FOR SEQ ID NO:89:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 16 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #AFM199xh12A) DESCRIPTION: Marker                                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:                                #    16            C                                                          - (2) INFORMATION FOR SEQ ID NO:90:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #AFM199xh12A) DESCRIPTION: Marker                                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:                                #   17             AG                                                         - (2) INFORMATION FOR SEQ ID NO:91:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #AFMa345wc9A) DESCRIPTION: Marker                                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:                                # 20               CACAT                                                      - (2) INFORMATION FOR SEQ ID NO:92:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #AFMa345wc9A) DESCRIPTION: Marker                                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Human                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:                                # 20               ACACA                                                      - (2) INFORMATION FOR SEQ ID NO:93:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (primer)                                        #for mouse Pax4 genePTION: primer                                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: murine                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:                                #                24GGTAC AAAG                                                 - (2) INFORMATION FOR SEQ ID NO:94:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 491 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                          (A) DESCRIPTION: Recombina - #nt murine met ob                      -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: murine                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 41..478                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:                                #CCG ATC CAG      55AACT TTTAGAAGGA GGAATAACAT ATG GTA                        #         Met Val Pro Ile Gln                                                 #       5  1                                                                  #AAA ACG ATC GTT ACG CGT     103CC TTA ATT                                    #Lys Thr Ile Val Thr Arg Lys Thr Leu Ile                                      #                 20                                                          #TCC GCT AAA CAG CGT GTT     151AG TCG GTC                                    #Ser Ala Lys Gln Arg Val Thr Gln Ser Val                                      #             35                                                              #CCG ATC CTA AGC TTG TCC     199GT CTG CAC                                    #Pro Ile Leu Ser Leu Ser Pro Gly Leu His                                      #         50                                                                  #CAG GTG TTA ACC TCC CTG     247TA TAC CAG                                    #Gln Val Leu Thr Ser Leu Ala Val Tyr Gln                                      #     65                                                                      #GAC CTC GAG AAC CTT CGC     295TC GCT AAC                                    #Asp Leu Glu Asn Leu Arg Gln Ile Ala Asn                                      # 85                                                                          #TCC TGC TCC CTG CCG CAG     343TC TCC AAA                                    #Ser Cys Ser Leu Pro Gln Ala Phe Ser Lys                                      #                100                                                          #GAC GGG GTC CTG GAA GCA     391AA TCC CTG                                    #Asp Gly Val Leu Glu Ala Pro Glu Ser Leu                                      #           115                                                               #TCC CGT CTG CAG GGT TCC     439TT GCT CTG                                    #Ser Arg Leu Gln Gly Ser Val Val Ala Leu                                      #       130                                                                   #TCT CCG GAA TGT TAATGGA     488TG GAC GTT                                    #Ser Pro Glu Cys Leu Gln Gln Leu Asp Val                                      #   145                                                                       #            491                                                              - (2) INFORMATION FOR SEQ ID NO:95:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 147 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                                       (A) DESCRIPTION: Recombina - #nt murine met ob protein              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:                                #Thr Lys Thr Leu Ile Lysys Val Gln Asp Asp                                    #                 15                                                          #His Thr Gln Ser Val Serle Asn Asp Ile Ser                                    #             30                                                              #Ile Pro Gly Leu His Prohr Gly Leu Asp Phe                                    #         45                                                                  #Leu Ala Val Tyr Gln Glnys Met Asp Gln Thr                                    #     60                                                                      #Leu Gln Ile Ala Asn Aspro Ser Gln Asn Val                                    # 80                                                                          #Leu Ala Phe Ser Lys Sersp Leu Leu His Leu                                    #                 95                                                          #Lys Pro Glu Ser Leu Asphr Ser Gly Leu Gln                                    #            110                                                              #Glu Val Val Ala Leu Serer Leu Tyr Ser Thr                                    #        125                                                                  #Gln Gln Leu Asp Val Sereu Gln Asp Ile Leu                                    #    140                                                                      -  Pro Glu Cys                                                                 145                                                                          - (2) INFORMATION FOR SEQ ID NO:96:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 454 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA                                                           (A) DESCRIPTION: Recombina - #nt human met ob                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: human                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 4..444                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:                                #GAC ACC AAA ACC TTA ATT      48TT CAG GAC                                         Met Val Pro Ile Gln Lys Val Gln - # Asp Asp Thr Lys Thr Leu Ile          #    15                                                                       #AGT CAC ACC CAG TCG GTG      96AC GAC ATC                                    #Ser His Thr Gln Ser Val Ile Asn Asp Ile                                      #30                                                                           #TTC ATC CCG GGT CTG CAC     144GC CTG GAC                                    #Phe Ile Pro Gly Leu His Thr Gly Leu Asp                                      #              45                                                             #ACC CTG GCT GTA TAC CAG     192TG GAC CAG                                    #Thr Leu Ala Val Tyr Gln Lys Met Asp Gln                                      #          60                                                                 #GTT CTT CAG ATC TCT AAC     240CC CGT AAC                                    #Val Leu Gln Ile Ser Asn Pro Ser Arg Asn                                      #      75                                                                     #GTG CTG GCA TTC TCC AAA     288TG CTG CAC                                    #Val Leu Ala Phe Ser Lys Asp Leu Leu His                                      #  95                                                                         #GAG ACT CTG GAC TCT CTG     336CA GGT CTT                                    #Glu Thr Leu Asp Ser Leu Ala Ser Gly Leu                                      #                110                                                          #ACC GAA GTT GTT GCT CTG     384GT TAC AGC                                    #Thr Glu Val Val Ala Leu Ser Gly Tyr Ser                                      #            125                                                              #CTT TGG CAG CTG GAC CTG     432AG GAC ATG                                    #Leu Trp Gln Leu Asp Leu Leu Gln Asp Met                                      #        140                                                                  #                454AATGGATCC                                                  Ser Pro Gly Cys                                                                   145                                                                      - (2) INFORMATION FOR SEQ ID NO:97:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 147 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                                       (A) DESCRIPTION: Recombina - #nt human met ob protein               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:                                #Thr Lys Thr Leu Ile Lysys Val Gln Asp Asp                                    #                 15                                                          #His Thr Gln Ser Val Serle Asn Asp Ile Ser                                    #              30                                                             #Ile Pro Gly Leu His Prohr Gly Leu Asp Phe                                    #          45                                                                 #Leu Ala Val Tyr Gln Glnys Met Asp Gln Thr                                    #      60                                                                     #Leu Gln Ile Ser Asn Aspro Ser Arg Asn Val                                    #  80                                                                         #Leu Ala Phe Ser Lys Sersp Leu Leu His Val                                    #95                                                                           #Thr Leu Asp Ser Leu Glyla Ser Gly Leu Glu                                    #            110                                                              #Glu Val Val Ala Leu Serer Gly Tyr Ser Thr                                    #        125                                                                  #Trp Gln Leu Asp Leu Sereu Gln Asp Met Leu                                    #    140                                                                      -  Pro Gly Cys                                                                 145                                                                          - (2) INFORMATION FOR SEQ ID NO:98:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 21 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: N-terminal                                          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:                                #Ser Ser Gly Leu Val Prois His His His His                                    #                 15                                                          -  Arg Gly Ser His Met                                                                     20                                                               - (2) INFORMATION FOR SEQ ID NO:99:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -      (v) FRAGMENT TYPE: N-terminal His-tag                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:                                #Ser Ser Gly Leu Val Prois His His His His                                    #                 15                                                          -  Arg Gly Ser Pro                                                                         20                                                               __________________________________________________________________________

What is claimed is:
 1. An antibody to a mammalian OB polypeptide, saidpolypeptide having the sequence of a naturally occurring mammalian OBpolypeptide, having as a mature protein about 145 amino acids, andcapable of modulating body weight.
 2. An antibody to an OB polypeptide,the polypeptide comprising:a) amino acid sequence set out in SEQ ID NO:2; b) the amino acid sequence set out in amino acids 22-167 of SEQ IDNO: 2; c) the amino acid sequence set out in SEQ ID NO: 4; or d) theamino acid sequence set out in amino acids 22-167 of SEQ ID NO:
 4. 3. Anantibody to an OB polypeptide, the polypeptide comprising:a) the aminoacid sequence set out in amino acids 22-167 of SEQ ID NO: 2 having anN-terminal methionine or an N-terminal polyhistidine; or b) the aminoacid sequence set out in amino acids 22-167 of SEQ ID NO: 4 having anN-terminal methionine or an N-terminal polyhistidine.
 4. An antibody toan OB polypeptide, the polypeptide comprising:a) the amino acid sequenceset out in SEQ ID NO: 5; b) the amino acid sequence set out in aminoacids 22-166 of SEQ ID NO: 5; c) the amino acid sequence set out inamino acids 22-166 of SEQ ID NO: 5, having an N-terminal methionine oran N-terminal polyhistidine; d) the amino acid sequence set out in SEQID NO: 6; e) the amino acid sequence set out in amino acids 22-166 ofSEQ ID NO: 6; or f) the amino acid sequence set out in amino acids22-166 of SEQ ID NO: 6 having an N-terminal methionine or an N-terminalpolyhistidine.
 5. An antibody to a variant of an OB polypeptidecomprising amino acids 22-167 of SEQ ID NO: 4 wherein one or more aminoacids selected from the group consisting of amino acids 53, 56, 71, 85,89, 92, 95, 98, 110, 118, 121, 122, 126, 127, 128, 129, 132, 139, 157,159, 163 and 166 is substituted with a conserved amino acid.
 6. Anantibody to a variant of an OB polypeptide comprising amino acids 22-167of SEQ ID NO: 4 wherein one or more amino acids selected from the groupconsisting of amino acids 53, 56, 71, 85, 89, 92, 95, 98, 110, 118, 121,122, 126, 127, 128, 129, 132, 139, 157, 159, 163 and 166 is substitutedwith the particular amino acid present at the corresponding position inSEQ ID NO:
 2. 7. An antibody to a variant of an OB polypeptidecomprising amino acids 22-166 of SEQ ID NO: 6 wherein one or more ofamino acids selected from the group consisting of amino acids 52, 55,70, 84, 88, 91, 94, 97, 109, 117, 120, 121, 125, 126, 127, 128, 131,138, 156, 158, 162 and 165 is substituted with a conserved amino acid.8. An antibody to a variant of an OB polypeptide comprising amino acids22-166 of SEQ ID NO: 6 wherein one or more of amino acids selected fromthe group consisting of amino acids 52, 55, 70, 84, 88, 91, 94, 97, 109,117, 120, 121, 125, 126, 127, 128, 131, 138, 156, 158, 162 and 165 issubstituted with the particular amino acid present at the correspondingposition in SEQ ID NO:
 5. 9. An antibody to an immunogenic fragment ofan OB polypeptide.
 10. An antibody to an immunogenic fragment of an OBpolypeptide selected from the group consisting of the polypeptide of SEQID NO: 18, the polypeptide of SEQ ID NO: 19, the polypeptide of SEQ IDNO: 20 and the polypeptide of SEQ ID NO:
 21. 11. An antibody to an OBpolypeptide analog wherein the OB polypeptide analog has 83 percent orgreater amino acid sequence identity to the OB polypeptide amino acidsequence set out in SEQ ID NOS: 2, 4, 5 or
 6. 12. An antibody accordingto any of claims 1-11 which is a polyclonal, monoclonal, chimeric orsingle chain antibody.
 13. An antibody according to any of claims 1-11which is an Fab, Fab', F(ab')₂ or F(v) portion of a whole antibodymolecule.
 14. An antibody according to claim 12 which is a humanizedmonoclonal antibody.
 15. An immortal cell line that produces amonoclonal antibody according to claim
 12. 16. A method for preparing anantibody specific to an OB polypeptide, comprising:(a) conjugating to acarrier protein a mammalian OB polypeptide, said polypeptide having thesequence of a naturally occurring mammalian OB polypeptide, having as amature protein about 145 amino acids, and capable of modulating bodyweight; (b) immunizing a host animal with the OB polypeptidefragment-carrier protein conjugate of step (a) admixed with an adjuvant;and (c) obtaining antibody from the immunized host animal.
 17. A methodfor preparing an antibody specific to an OB polypeptide, comprising:(a)conjugating to a carrier protein an OB polypeptide comprising:i) theamino acid sequence set out in SEQ ID NO: 2; ii) the amino acid sequenceset out in amino acids 22-167 of SEQ ID NO: 2; iii) the amino acidsequence set out in SEQ ID NO: 4; or iv) the amino acid sequence set outin amino acids 22-167 of SEQ ID NO: 4; (b) immunizing a host animal withthe OB polypeptide fragment-carrier protein conjugate of step (a)admixed with an adjuvant; and (c) obtaining antibody from the immunizedhost animal.
 18. A method for preparing an antibody specific to an OBpolypeptide, comprising:(a) conjugating to a carrier protein an OBpolypeptide comprising:i) the amino acid sequence set out in SEQ ID NO:5; ii) the amino acid sequence set out in amino acids 22-166 of SEQ IDNO: 5; iii) the amino acid sequence set out in SEQ ID NO: 6; or iv) theamino acid sequence set out in amino acids 22-166 of SEQ ID NO: 6; (b)immunizing a host animal with the OB polypeptide fragment-carrierprotein conjugate of step (a) admixed with an adjuvant; and (c)obtaining antibody from the immunized host animal.
 19. A method forpreparing an antibody specific to an OB polypeptide, comprising:(a)conjugating to a carrier protein an immunogenic fragment of an OBpolypeptide; (b) immunizing a host animal with the OB polypeptidefragment-carrier protein conjugate of step (a) admixed with an adjuvant;and (c) obtaining antibody from the immunized host animal.
 20. Themethod of according to claim wherein the immunogenic fragment isselected from the group of the polypeptide of SEQ ID NO: 18, thepolypeptide of SEQ ID NO: 19, the polypeptide of SEQ ID NO: 20, and thepolypeptide of SEQ ID NO:
 21. 21. A method for preparing an antibodyspecific to an OB polypeptide, comprising:(a) conjugating to a carrierprotein an OB polypeptide comprising amino acids 22-167 of SEQ ID NO: 4wherein one or more amino acids selected from the group consisting ofamino acids 53, 56, 71, 85, 89, 92, 95, 98, 110, 118, 121, 122, 126,127, 128, 129, 132, 139, 157, 159, 163 and 166 is substituted with aconserved amino acid; (b) immunizing a host animal with the OBpolypeptide fragment-carrier protein conjugate of step (a) admixed withan adjuvant; and (c) obtaining antibody from the immunized host animal.22. A method for preparing an antibody specific to an OB polypeptide,comprising:(a) conjugating to a carrier protein an OB polypeptidecomprising amino acids 22-167 of SEQ ID NO: 4 wherein one or more aminoacids selected from the group consisting of amino acids 53, 56, 71, 85,89, 92, 95, 98, 110, 118, 121, 122, 126, 127, 128, 129, 132, 139, 157,159, 163 and 166 is substituted with the particular amino acid presentat the corresponding position in SEQ ID NO: 2; (b) immunizing a hostanimal with the OB polypeptide fragment-carrier protein conjugate ofstep (a) admixed with an adjuvant; and (c) obtaining antibody from theimmunized host animal.
 23. A method for preparing an antibody specificto an OB polypeptide, comprising:(a) conjugating to a carrier protein anOB polypeptide having 83 percent or greater amino acid sequence identityto the OB polypeptide amino acid sequence set out in SEQ ID NOS: 2, 4, 5or 6; (b) immunizing a host animal with the OB polypeptidefragment-carrier protein conjugate of step (a) admixed with an adjuvant;and (c) obtaining antibody from the immunized host animal.
 24. A testkit for determining the presence or amount of OB polypeptidecomprising:(a) a predetermined amount of at least one labeled antibodyto an OB polypeptide obtained by the direct or indirect attachment of anantibody to an OB polypeptide to a detectable label; (b) other reagents;and (c) directions for use of said kit.
 25. A test kit for determiningthe presence or amount of OB polypeptide comprising:(a) a labeledcomponent which has been obtained by coupling an OB polypeptide to adetectable label; (b) one or more additional immunochemically reactivecomponents of which at least one component is an antibody to an OBpolypeptide; and (c) directions for the performance of a protocol forthe detection and/or determination of one or more components of animmunochemical reaction between OB polypeptide and an antibody to an OBpolypeptide.
 26. A test kit of claim 24 or 25, wherein the kitdetermines the presence or amount of OB polypeptide in an individual'sblood or plasma.
 27. A test kit of claim 24 or 25, wherein thedetectable label is radioactive, enzymatic, fluorescent or calorimetric.